Methods relating to peripheral administration of nogo receptor polypeptides

ABSTRACT

This invention relates to methods of treating diseases involving accumulation of Aβ plaques, including Alzheimer&#39;s Disease by the peripheral administration of soluble Nogo receptor polypeptides. The invention also provides methods of increasing the plasma to brain ratio of Aβ peptide and enhancing Aβ peptide clearance via peripheral administration of soluble Nogo receptor polypeptides. This invention also provides methods of improving memory function or inhibiting memory loss via the peripheral administration of soluble Nogo receptor polypeptides. The invention also provides methods of decreasing the size and number of Aβ plaques in a mammal via peripheral administration of soluble Nogo receptor polypeptides.

FIELD OF THE INVENTION

This invention relates to, neurobiology, neurology and pharmacology.More particularly, this invention relates to methods of treatingdiseases involving Aβ plaque accumulation, including Alzheimer's Diseaseby the peripheral administration of soluble Nogo receptor polypeptides.The invention also provides methods of increasing the plasma to brainratio of Aβ peptide and enhancing Aβ peptide clearance via peripheraladministration of soluble Nogo receptor polypeptides. This inventionalso provides methods of improving memory function or inhibiting memoryloss via the peripheral administration of soluble Nogo receptorpolypeptides. The invention further provides methods of reducing Aβplaque size and Aβ plaque number via peripheral administration ofsoluble Nogo receptor polypeptides.

BACKGROUND OF THE INVENTION

Neurodegeneration in Alzheimer's Disease (AD) is accompanied by amyloidplaques and neurofibrillary tangles. Glenner et al., Science 297:353-356(2002). The amyloid plaques are composed primarily of a 40-43 aa Amyloidβ (Aβ) peptide that derives from proteolytic cleavage of amyloidprecursor protein (APP). Li et al., Proc Acad Sci USA 92:12180-12184(1995); Sinha et al., Nature 402:537-540 (1999); Vassar et al., Science286:735-741, (1999). Potential therapies include decreasing Aβproduction (Lanz et al., J Pharmacol Exp Ther 305:864-871 (2003)) withsecretase inhibitors, increasing Aβ degradation (Frautschy et al., Am JPathol 140:1389-1399 (1992)) with zinc metalloendopeptidases such asinsulin-degrading enzyme (IDE) (Qiu et al., J Biol Chem 273:32730-32738(1998); Bertram et al., Science 290:2302-2303 (2000)) or neprilysin(NEP) (Yasojima et al., Neurosci Lett 297:97-100 (2001); Iwata et al.,Science 292:1550-1552 (2001)), and promoting Ab-specific immunity(Younkin S G, Nat Med 7:18-19 (2001); Morgan et al., Nature 408:982-985(2000); Lee V M. Proc Natl Acad Sci USA 98:8931-8932 (2001)). However,problems with toxicity and clearing the blood-brain barrier (BBB) havehampered efforts to treat AD. Birmingham K. and Frantz S. Nat Med8:199-200 (2002) and Orgogozo et al., Neurology 61:46-54 (2003).

The Nogo-66 receptor (NgR1) participates in limiting injury-inducedaxonal growth and experience-dependent plasticity in the adult brain.Fournier et al., Nature 409:341-346 (2001); McGee A. W. and StrittmatterS. M. Trends Neurosci 26:193-198 (2003); McGee et al., Science309:2222-2226 (2005). See also PCT Publication Nos. WO 2005/016955, WO03/031462, WO 2004/014311, and WO 01/51520, as well as U.S. PatentPublications US 2002-0077295 and US 2005-0271655 A1, all of which areincorporated herein by reference in their entireties.

In this role, it serves as a receptor for three myelin inhibitorproteins, Nogo, MAG and OMgp, signaling to activate Rho GTPase in axons.Fournier et al., Nature 409:341-346 (2001); Liu et al., Science297:1190-1193 (2002); Wang et al.; Nature 417:941-944 (2002); Fournieret al., J Neurosci 23; 1416-1423 (2003); McGee A. W. and Strittmatter S.M. Trends Neurosci 26:193-198 (2003). In addition, brain NgR1 interactswith APP through its Aβ domain. Park et al., J Neurosci 26:1386-1395(2006). Moreover, increased levels of brain NgR1 result in reduced Aβload, while loss of endogenous NgR1 elevates Aβ. Parallel changes in Aβand secreted APPα plus APPβ suggest that at least a portion of the invivo effects of brain NgR1 on Aβ levels is mediated by blockade ofα/β-secretase activity. However, the high affinity of NgR1 for Aβ andthe presence of NgR1 in plaques imply that NgR1 might also regulate theclearance of Aβ. Park et al., J Neurosci 26:1386-1395 (2006).

Immunological methods have been successful in decreasing Aβ plaqueburden, as reviewed by Schenk. Schenk D. Nat Rev Neurosci 3:824-828(2002). Both active and passive immunizations have promoted efflux,inhibited influx, or activated microglia-induced Ab degradation. WeinerH. L. and Selkoe D. J. Nature 420:879-884 (2002); Morgan et al., Nature408:982-985 (2000); Schenk et al., Nature 400:173-177 (1999). Activeimmunization with Aβ1-42 plus adjuvant in PD-mAPP reduced Aβ plaquepathology. Schenk et al., Nature 400:173-177 (1999). Bard et al.demonstrated that humoral immunity is sufficient to reduce plaque burdenby triggering antibody trafficking across the blood brain barrier. Bardet al., Nat Med 6:916-919 (2000). In contrast, DeMattos et al.,demonstrated that an Aβ antibody reduces Alzheimer pathology withoutantibody passage across the BBB, implicating a peripheral sink mechanismfor anti-Aβ reductions in Alzheimer's pathology. DeMattos et al., ProcNatl Acad Sci USA 98:8850-8855 (2001).

In a range of studies, reducing Aβ burden in brain by immunologicalmeans has been associated with improved spatial memory performance inAlzheimer model transgenic mice. However, in several reports, behavioralimprovements occurred acutely, prior to any change in plaque density,suggesting the antibody association with particular soluble Aβ speciesis responsible for improved function. Two non-immunoglobulin proteins,RAGE and gelsolin, have been shown to bind Aβ and, when administeredperipherally, to decrease brain Aβ load. Deane et al., Nat Med 9:907-913(2003); Matsuoka et al., J Neurosci 23:29-33 (2003); Arancio et al.,Embo J 23:4096-4105 (2004). Whether Aβ reduction by peripheralnon-antibody Aβ-binding proteins is associated with improved cognitiveand memory function has not been tested.

SUMMARY OF THE INVENTION

This invention is based on the discovery that the administration ofsoluble NgR1 polypeptides peripheral to the central nervous systemenhanced Aβ clearance from the brain and improved memory function.

In certain embodiments, the invention includes a method for increasingthe plasma to brain ratio of Aβ peptide in a mammal, comprisingadministering a therapeutically effective amount of a soluble Nogoreceptor polypeptide, wherein said administration is peripheral to thecentral nervous system.

In certain embodiments, the invention includes a method for enhancing Aβclearance from the brain of a mammal, comprising administering atherapeutically effective amount of a soluble Nogo receptor polypeptide,wherein said administration is peripheral to the central nervous system.

In certain embodiments, the invention includes a method for improvingmemory function or inhibiting memory loss in a mammal comprisingadministering a therapeutically effective amount of a soluble Nogoreceptor polypeptide, wherein said administration is peripheral to thecentral nervous system.

In certain embodiments, the invention provides a method of reducing thenumber of Aβ plaques in the brain of a mammal, comprising administeringto a mammal in need thereof a therapeutically effective amount of asoluble Nogo receptor polypeptide, wherein said administration isperipheral to the central nervous system.

In certain embodiments, the invention provides a method of reducing thesize of Aβ plaques in the brain of a mammal, comprising administering toa mammal in need thereof a therapeutically effective amount of a solubleNogo receptor polypeptide, wherein said administration is peripheral tothe central nervous system.

In certain embodiments, the invention provides a method of treating adisease associated with Aβ plaque accumulation in a mammal comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of a soluble Nogo receptor polypeptide, wherein saidadministration is peripheral to the central nervous system. In someembodiments, the disease is selected from the group consisting ofAlzheimer's disease, mild cognitive impairment, mild-to-moderatecognitive impairment, vascular dementia, cerebral amyloid angiopathy,hereditary cerebral hemorrhage, senile dementia, Down's syndrome,inclusion body myositis, age-related macular degeneration, primaryamyloidosis, secondary amyloidosis and a condition associated withAlzheimer's disease. In some embodiments, the condition associated withAlzheimer's disease is selected from the group consisting ofhypothyroidism, cerebrovascular disease, cardiovascular disease, memoryloss, anxiety, a behavioral dysfunction, a neurological condition, and apsychological condition. In some embodiments, the behavioral dysfunctionis selected from the group consisting of apathy, aggression, andincontinence. In some embodiments, the neurological condition isselected from the group consisting of Huntington's disease, amyotrophiclateral sclerosis, acquired immunodeficiency, Parkinson's disease,aphasia, apraxia, agnosia, Pick disease, dementia with Lewy bodies,altered muscle tone, seizures, sensory loss, visual field deficits,incoordination, gait disturbance, transient ischemic attack or stroke,transient alertness, attention deficit, frequent falls, syncope,neuroleptic sensitivity, normal pressure hydrocephalus, subduralhematoma, brain tumor, posttraumatic brain injury, and posthypoxicdamage. In some embodiments, the psychological condition is selectedfrom the group consisitng of depression, delusions, illusions,hallucinations, sexual disorders, weight loss, psychosis, a sleepdisturbance, insomnia, behavioral disinhibition, poor insight, suicidalideation, depressed mood, irritability, anhedonia, social withdrawal,and excessive guilt. In one embodiment, the mammal is a human.

In some embodiments, the soluble Nogo receptor polypeptide isadministered subcutaneously, parenteraly, intravenously,intramuscularly, intraperitoneally, transdermally, inhalationaly orbuccally. In one embodiment, the soluble Nogo receptor polypeptide isadministered subcutaneously.

In some embodiments, the soluble Nogo receptor polypeptide is 90%identical to a reference amino acid sequence is selected from the groupconsisting of: (i) amino acids 27 to 310 of SEQ ID NO:2; (ii) aminoacids 27 to 344 of SEQ ID NO:2; (iii) amino acids 27 to 445 of SEQ IDNO:2; (iv) amino acids 27 to 309 of SEQ ID NO:2; (v) amino acids 1 to310 of SEQ ID NO:2; (vi) amino acids 1 to 344 of SEQ ID NO:2; (vii)amino acids 1 to 445 of SEQ ID NO:2; (viii) amino acids 1 to 309 of SEQID NO:2; (ix) variants or derivatives of any of said reference aminoacid sequences, and (x) a combination of one or more of said referenceamino acid sequences or variants or derivatives thereof.

In some embodiments, the soluble NgR1 polypeptide is selected from thegroup consisting of: (i) amino acids 27 to 310 of SEQ ID NO:2; (ii)amino acids 27 to 344 of SEQ ID NO:2; (iii) amino acids 27 to 445 of SEQID NO:2; (iv) amino acids 27 to 309 of SEQ ID NO:2; (v) amino acids 1 to310 of SEQ ID NO:2; (vi) amino acids 1 to 344 of SEQ IIS NO:2; (vii)amino acids 1 to 445 of SEQ ID NO:2; (viii) amino acids 1 to 309 of SEQID NO:2; (ix) variants or derivatives of any of said polypeptides; and(x) a combination of one or more of said polypeptides or variants orderivatives thereof. In one embodiment, the soluble Nogo receptorpolypeptide comprises amino acids 27 to 310 of SEQ ID NO:2. In oneembodiment, the soluble Nogo receptor polypeptide comprises amino acids27 to 344 of SEQ ID NO:2. In one embodiment, the soluble Nogo receptorpolypeptide comprises amino acids 27 to 445 of SEQ ID NO:2. In oneembodiment, the soluble Nogo receptor polypeptide comprises amino acids27 to 309 of SEQ ID NO:2. In one embodiment, the soluble Nogo receptorpolypeptide comprises amino acids 1 to 310 of SEQ ID NO:2. In oneembodiment, the soluble Nogo receptor polypeptide comprises amino acids1 to 344 of SEQ ID NO:2. In one embodiment, the soluble Nogo receptorpolypeptide comprises amino acids 1 to 445 of SEQ ID NO:2. In oneembodiment, the soluble Nogo receptor polypeptide comprises amino acids1 to 309 of SEQ ID NO:2.

In some embodiments, the soluble Nogo receptor polypeptide comprises afirst polypeptide fragment and a second polypeptide fragment, whereinsaid first polypeptide fragment comprises an amino acid sequenceidentical to a first reference amino acid sequence, except for up totwenty individual amino acid substitutions, wherein said first referenceamino acid sequence is selected from the group consisting of (a) aminoacids a to 445 of SEQ ID NO:2, (b) amino acids 27 to b of SEQ ID NO:2,and (c) amino acids a to b of SEQ ID NO:2, wherein a is any integer from25 to 35, and b is any integer from 300 to 450; wherein said secondpolypeptide fragment comprises an amino acid sequence identical to asecond reference amino acid sequence, except for up to twenty individualamino acid substitutions, wherein said second reference amino acidsequence is selected from the group consisting of (a) amino acids c to445 of SEQ ID NO:2, (b) amino acids 27 to d of SEQ ID NO:2, and (c)amino acids c to d of SEQ ID NO:2, wherein c is any integer from 25 to35, and d is any integer from 300 to 450. In some embodiments, the firstpolypeptide fragment is situated upstream of said second polypeptidefragment. In a further embodiment, a peptide linker is situated betweenthe first polypeptide fragment and the second polypeptide fragment. Inone embodiment, the peptide linker comprises SEQ ID NO:18 (G4S)₃.

In some embodiments, at least one amino acid residue of the soluble NgR1polypeptide is substituted with a different amino acid. In someembodiments, the different amino acid is selected from the groupconsisting of alanine, serine and threonine. In one embodiment, thedifferent amino acid is alanine.

In some embodiments, the soluble NgR polypeptides are cyclic. In someembodiments, the cyclic polypeptides further comprise a first moleculelinked at the N-terminus and a second molecule linked at the C-terminus;wherein the first molecule and the second molecule are joined to eachother to form said cyclic molecule. In some embodiments, the first andsecond molecules are selected from the group consisting of: a biotinmolecule, a cysteine residue, and an acetylated cysteine residue. Insome embodiments, the first molecule is a biotin molecule attached tothe N-terminus and the second molecule is a cysteine residue attached tothe C-terminus of the polypeptide of the invention. In some embodiments,the first molecule is an acetylated cysteine residue attached to theN-terminus and the second molecule is a cysteine residue attached to theC-terminus of the polypeptide of the invention. In some embodiments, thefirst molecule is an acetylated cysteine residue attached to theN-terminus and the second molecule is a cysteine residue attached to theC-terminus of the polypeptide of the invention. In some embodiments, theC-terminal cysteine has an NH2 moiety attached.

In some embodiments, the soluble NgR1 polypeptide further comprises anon-NgR1 moiety. In some embodiments, the non-NgR1 moiety is aheterologous polypeptide fused to the soluble NgR1 polypeptide. In someembodiments, the invention further provides that the heterologouspolypeptide is selected from the group consisting of: (a) serum albumin,(b) an Fc region, (c) a signal peptide, (d) a polypeptide tag, and (e) acombination of two or more of said heterologous polypeptides. In someembodiments, the invention further provides that the Fc region isselected from the group consisting of an IgA Fc region; an IgD Fcregion; an IgG Fc region, an IgEFc region; and an IgM Fc region. In oneembodiment, the Fc region is an IgG Fc region. In some embodiments, theinvention further provides that a peptide linker is situated between theamino acid sequence and the IgG Fc region. In one embodiment, thepeptide linker comprises SEQ ID NO:19(G4S)₂. In some embodiments, theinvention further provides that the polypeptide tag is selected from thegroup consisting of: FLAG tag; Strep tag; poly-histidine tag; VSV-G tag;influenza virus hemagglutinin (HA) tag; and c-Myc tag.

In some embodiments, the invention provides a polypeptide of theinvention attached to one or more polyalkylene glycol moieties. In someembodiments, the invention further provides that the one or morepolyalkylene glycol moieties is a polyethylene glycol (PEG) moiety. Insome embodiments, the invention further provides a polypeptide of theinvention attached to 1 to 5 PEG moieties.

In some embodiments, the invention provides that the therapeuticallyeffective amount is from 0.001 mg/kg to 10 mg/kg of soluble Nogoreceptor polypeptide. In some embodiments, the therapeutically effectiveamount is from 0.01 mg/kg to 1 mg/kg of soluble Nogo receptorpolypeptide. In some embodiments, the therapeutically effective amountis from 0.05 mg/kg to 0.5 mg/kg of soluble Nogo receptor polypeptide.

In some embodiments, the invention provides that the soluble Nogoreceptor polypeptide does not cross the blood-brain bather.

In some embodiments, the soluble Nogo receptor polypeptide iscoadministered with one or more anti-Aβ antibodies. In some embodiments,the soluble Nogo receptor polypeptide is coadministered with one or moreadditional therapeutic agents, selected from the group consisting of anadrenergic agent, anti-adrenergic agent, anti-androgen agent,anti-anginal agent, anti-anxiety agent, anticonvulsant agent,antidepressant agent, anti-epileptic agent, antihyperlipidemic agent,antihyperlipoproteinemic agent, antihypertensive agent,anti-inflammatory agent, antiobessional agent, antiparkinsonian agent,antipsychotic agent, adrenocortical steroid; adrenocortical suppressant;aldosterone antagonist; amino acid; anabolic steroid; analeptic agent;androgen; blood glucose regulator; cardioprotectant agent;cardiovascular agent; cholinergic agonist or antagonist; cholinesterasedeactivator or inhibitor, cognition adjuvant or enhancer; dopaminergicagent; enzyme inhibitor, estrogen, free oxygen radical scavenger; GABAagonist; glutamate antagonist; hormone; hypocholesterolemic agent;hypolipidemic agent; hypotensive agent; immunizing agent;immunostimulant agent; monoamine oxidase inhibitor, neuroprotectiveagent; NMDA antagonist; AMPA antagonist, competitive or -non-competitiveNMDA antagonist; opioid antagonist; potassium channel opener;non-hormonal sterol derivative; post-stroke and post-head traumatreatment; prostaglandin agent; psychotropic agent; relaxant agent;sedative agent; sedative-hypnotic agent; selective adenosine antagonist;serotonin antagonist; serotonin inhibitor; selective serotonin uptakeinhibitor; serotonin receptor antagonist; sodium and calcium channelblocker; steroid; stimulant; and thyroid hormone and inhibitor agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows deletion mapping of the AP-Aβ(1-28) region required forbinding to COS-7 cells expressing wild type NgR1. FIG. 1B shows thatAP-Aβ(1-28) does not bind to COS-7 cells expressing p75-NTR or RAGEunder conditions that allow binding to cells expressing NgR1. FIG. 1Cshows displacement of AP-Aβ(1-28) but not AP-Nogo-66(1-33), from NgR1 byAβ(1-28).

FIG. 2A shows mutant NgR1 proteins at the surface of transfected COS-7cells were detected by immunostaining with rabbit anti-NgR1 antibodyrecognized by anti-rabbit-AP. FIG. 2B shows binding of AP or AP fusedNgR1 ligands to COS-7 cells expressing NgR1 mutants displayingdifferential binding. FIG. 2C depicts cell lysate of COS-7 cellsexpressing NgR1 and mutants that were immunoblotted with anti-NgR1antibody to ascertain molecular weight and expression levels. FIG. 2Dshows quantification of AP binding of NgR1 ligands to NgR1 mutantsexpressed as a percentage of wild type NgR1.

FIG. 3A shows an anti-NgR1 immunoblot of protein concentrated by proteinA/G affinity chromotography in brain lysate of APPswe/PSEN-1ΔE9transgenic mice treated subcutaneously with rat IgG, subcutaneously withNgR1(310)ecto-Fc or i.c.v. with NgR1(310)ecto-Fc. FIG. 3B shows ratio ofplasma versus brain Aβ level in peripherally-treated Appswe/PSEN-1ΔE9transgenic mice at 10 months of age plotted as a percentage. FIG. 3Cshows an anti-APP (6E10) Immunoblot of brain lysate of APPswe/PSEN-1ΔE9transgenic mice treated subcutaneously with rat IgG or subcutaneouslywith NgR1(310)ecto-Fc from 7-10 months of age. FIG. 3D shows the levelof anti-APP immunoreactivity in brain lysates.

FIG. 4A shows examples of anti-Aβ immunoreactive plaque deposits incerebral cortex of control and NgR1(310)ecto-Fc treated transgenic mice.FIG. 4B shows examples of anti-synaptophysin immunoreactive plaquedeposits in hippocampus. FIG. 4C shows anti-GFAP immunoreactivity in thehippocampus. FIG. 4D shows the percentage of area occupied by Aβ plaquequantified from images in FIG. 4A. FIG. 4E shows Aβ(1-40) and Aβ(1-42)levels assessed by ELISA between NgR1(310)ecto-Fc and rat IgG groups.FIG. 4F shows the area occupied by anti-synaptophysin immunoreactivedystrophic neurites from FIG. 4B. FIG. 4G shows the percentage of areaoccupied by anti-GFAP immunoreactivity as measured from images in FIG.4C.

FIG. 5A shows the average number of errors in a six-arm radial watermaze for APPswe/PSEN-1ΔE9 and wild type litter mates at 4 months of age.FIG. 5B shows the average number of errors in a six-arm radial watermaze for APPswe/PSEN-1ΔE9 and wild type litter mates at 13 months ofage. FIG. 5C shows the average number of errors in a six-arm radialwater maze for NgR+/− and NgR−/− mice. FIG. 5D shows the results fromsubcutaneous treatment of NgR1(310)ecto-Fc in APPswe/PSEN-1ΔE9 mice frommonths 7-10. FIG. 5E shows a scatter plot between plaque density andaverage errors per swim for the last ten trials for each mouse.

FIG. 6 shows the visible platform escape latency of APPswe/PSEN-1ΔE9transgenic mice after subcutaneous treatment with NgR1(310)ecto-Fc orIgG from age 7 months to 10 months.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Also, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes.

Although methods and materials similar or equivalent to those describedherein can be used in practice or testing of the present invention,suitable methods and materials are described below. The materials,methods and examples are illustrative only, and are not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

In order to further define this invention, the following terms anddefinitions are herein provided.

It is to be noted that the term “a” or “an” entity, refers to one ormore of that entity; for example, “an immunoglobulin molecule,” isunderstood to represent one or more immunoglobulin molecules. As such,the terms “a” (or “an”), “one or more,” and “at least one” can be usedinterchangeably herein.

As used herein, the term “consists of,” or variations such as “consistof” or “consisting of,” as used throughout the specification and claims,indicate the inclusion of any recited integer or group of integers, butthat no additional integer or group of integers may be added to thespecified method, structure or composition.

As used herein, the term “consists essentially of,” or variations suchas “consist essentially of” or “consisting essentially of,” as usedthroughout the specification and claims, indicate the inclusion of anyrecited integer or group of integers, and the optional inclusion of anyrecited integer or group of integers that do not materially change thebasic or novel properties of the specified method, structure orcomposition.

As used herein, “antibody” means an intact immunoglobulin, or anantigen-binding fragment thereof. Antibodies of this invention can be ofany isotype or class (e.g., M, D, G, E and A) or any subclass (e.g.,G1-4, A1-2) and can have either a kappa (κ) or lambda (λ) light chain.

As used herein, “Fc” means a portion of an immunoglobulin heavy chainthat comprises one or more heavy chain constant region domains, CH1, CH2and CH3. For example, a portion of the heavy chain constant region of anantibody that is obtainable by papain digestion.

As used herein and in U.S. patent application 60/402,866, “Nogoreceptor,” “NogoR,”, “NogoR-1,” “NgR,” “NgR-1,” “NgR1” and “NGR1” eachmeans Nogo receptor-1.

As used herein, “NogoR fusion protein” means a protein comprising asoluble Nogo receptor-1 moiety fused to a heterologous polypeptide.

As used herein, “humanized antibody” means an antibody in which at leasta portion of the non-human sequences are replaced with human sequences.Examples of how to make humanized antibodies may be found in U.S. Pat.Nos. 6,054,297, 5,886,152 and 5,877,293.

As used herein, “chimeric antibody” means an antibody that contains oneor more regions from a first antibody and one or more regions from atleast one other antibody. The first antibody and the additionalantibodies can be from the same or different species.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

In the present invention, a “polypeptide fragment” refers to a shortamino acid sequence of a larger polypeptide. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part of region. Representative examples of polypeptidefragments of the invention, include, for example, fragments comprisingabout 5 amino acids, about 10 amino acids, about 15 amino acids, about20 amino acids, about 30 amino acids, about 40 amino acids, about 50amino acids, about 60 amino acids, about 70 amino acids, about 80 aminoacids, about 90 amino acids, and about 100 amino acids or more inlength.

The terms “fragment,” “variant,” “derivative” and “analog” whenreferring to a polypeptide of the present invention include anypolypeptide which retains at least some biological activity.Polypeptides as described herein may include fragment, variant, orderivative molecules therein without limitation, so long as thepolypeptide still serves its function. NgR1 polypeptides and polypeptidefragments of the present invention may include proteolytic fragments,deletion fragments and in particular, fragments which more easily reachthe site of action when delivered to an animal. Polypeptide fragmentsfurther include any portion of the polypeptide which comprises anantigenic or immunogenic epitope of the native polypeptide, includinglinear as well as three-dimensional epitopes. NgR1 polypeptides andpolypeptide fragments of the present invention may comprise variantregions, including fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally, such as anallelic variant. By an “allelic variant” is intended alternate forms ofa gene occupying a given locus on a chromosome of an organism. Genes II,Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturallyoccurring variants may be produced using art-known mutagenesistechniques. NgR1 polypeptides and polypeptide fragments of the inventionmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. NgR1 polypeptides and polypeptide fragments ofthe present invention may also include derivative molecules. Variantpolypeptides may also be referred to herein as “polypeptide analogs.” Asused herein a “derivative” of a polypeptide or a polypeptide fragmentrefers to a subject polypeptide having one or more residues chemicallyderivatized by reaction of a functional side group. Also included as“derivatives” are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup.

As used herein, “fusion protein” means a protein comprising a firstpolypeptide linearly connected, via peptide bonds, to a second,polypeptide. The first polypeptide and the second polypeptide may beidentical or different, and they may be directly connected, or connectedvia a peptide linker (see below).

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide can contain the nucleotide sequence of thefull-length cDNA sequence, including the untranslated 5′ and 3′sequences, the coding sequences. A polynucleotide may comprise aconventional phosphodiester bond or a non-conventional bond (e.g., anamide bond, such as found in peptide nucleic acids (PNA)). Thepolynucleotide can be composed of any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. For example, polynucleotides can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the polynucleotides can be composed of triple-stranded regionscomprising RNA or DNA or both RNA and DNA. polynucleotides may alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

The term “nucleic acid” refer to any one or more nucleic acid segments,e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated”nucleic acid or polynucleotide is intended a nucleic acid molecule, DNAor RNA, which has been removed from its native environment. For example,a recombinant polynucleotide encoding an NgR1 polypeptide or polypeptidefragment of the invention contained in a vector is considered isolatedfor the purposes of the present invention. Further examples of anisolated polynucleotide include recombinant polynucleotides maintainedin heterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding anNgR1 polypeptide or polypeptide fragment of the present invention.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” and “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two ore more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature.) Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence.

A “linker” sequence is a series of one or more amino acids separatingtwo polypeptide coding regions in a fusion protein. A typical linkercomprises at least 5 amino acids. Additional linkers comprise at least10 or at least 15 amino acids. In certain embodiments, the amino acidsof a peptide linker are selected so that the linker is hydrophilic. Thelinker (Gly-Gly-Gly-Gly-Ser)₃ (G4S)₃ (SEQ ID NO:18) is a preferredlinker that is widely applicable to many antibodies as it providessufficient flexibility. Other linkers include (Gly-Gly-Gly-Gly-Ser)₂(G4S)₂ (SEQ ID NO:19), Glu Ser Gly Arg Ser Gly Gly Gly Gly Ser Gly GlyGly Gly Ser (SEQ ID NO:20), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu SerLys Ser Thr (SEQ ID NO:21), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu SerLys Ser Thr Gln (SEQ ID NO:22), Glu Gly Lys Ser Ser Gly Ser Gly Ser GluSer Lys Val Asp (SEQ ID NO:23), Gly Ser Thr Ser Gly Ser Gly Lys Ser SerGlu Gly Lys Gly (SEQ ID NO:24), Lys Glu Ser Gly Ser Val Ser Ser Glu GlnLeu Ala Gln Phe Arg Ser Leu Asp (SEQ ID NO:25), and Glu Ser Gly Ser ValSer Ser Glu Glu Leu Ala Phe Arg Ser Leu Asp (SEQ ID NO:26). Examples ofshorter linkers include fragments of the above linkers, and examples oflonger linkers include combinations of the linkers above, combinationsof fragments of the linkers above, and combinations of the linkers abovewith fragments of the linkers above.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes, withoutlimitation, transcription of the gene into messenger RNA (mRNA),transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA(siRNA) or any other RNA product, and the translation of such mRNA intopolypeptide(s), as well as any processes which regulate eithertranscription or translation. If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of multiplesclerosis. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include, but arenot limited to, humans, domestic animals, farm animals, zoo animals,sport animals, pet animals such as dogs, cats, guinea pigs, rabbits,rats, mice, horses, cattle, cows; primates such as apes, monkeys,orangutans, and chimpanzees; canids such as dogs and wolves; felids suchas cats, lions, and tigers; equids such as horses, donkeys, and zebras;food animals such as cows, pigs, and sheep; ungulates such as deer andgiraffes; rodents such as mice, rats, hamsters and guinea pigs; and soon. In certain embodiments, the mammal is a human subject.

As used herein, a “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutic result may be, e.g., lesseningof symptoms, prolonged survival, improved mobility, and the like. Atherapeutic result need not be a “cure”.

As used herein, a “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

As used herein, “Aβ clearance” refers to a shift of Aβ peptide from thebrain to the plasma.

Nogo Receptor-1 Polypeptides

The human NgR1 polypeptide is shown below as SEQ ID NO:2.

Full-Length Human NgR1 (SEQ ID NO: 2):MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEPKVTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPAASFRACRNLTILWLHSNVLARIDAAAFTGLALLEQLDLSDNAQLRSVDPATFHGLGRLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNALQALPDDTFRDLGNLTHLFLHGNRISSVPERAFRGLHSLDRLLLHQNRVAHVHPHAFRDLGRLMTLYLFANNLSALPTEALAPLRALQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCSLPQRLAGRDLKRLAANDLQGCAVATGPYHPIWTGRATDEEPLGLPKCCQPDAADKASVLEPGRPASAGNALKGRVPPGDSPPGNGSGPRHINDSPFGTLPGSAEPPLTAVRPEGSEPPGFPTSGPRRRPGCSRKNRTRSHCRLGQAGSGGGGTGDSEGSGALPSLTCSLTPLGLALVLWTVLGPC

The rat NgR1 polypeptide is shown below as SEQ ID NO:4.

Full-Length Rat NgR1 (SEQ ID NO: 4):MKRASSGGSRLLAWVLWLQAWRVATPCPGACVCYNEPKVTTSCPQQGLQAVPTGIPASSQRIFLHGNRISHVPAASFQSCRNLTILWLHSNALARIDAAAFTGLTLLEQLDLSDNAQLHVVDPTTFHGLGHLHTLHLDRCGLRELGPGLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFLHGNRIPSVPEHAFRGLHSLDRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLMPLRSLQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCNLPQRLADRDLKRLAASDLEGCAVASGPFRPIQTSQLTDEELLSLPKCCQPDAADKASVLEPGRPASAGNALKGRVPPGDTPPGNGSGPRHINDSPFGTLPSSAEPPLTALRPGGSEPPGLPTTGPRRRPGCSRKNRTRSHCRLGQAGSGASGTGDAEGSGALPALACSLAPLGLALVLWTVLGPC

The mouse NgR1 polypeptide is shown below as SEQ ID NO:6.

Full-Length Mouse NgR1 (SEQ ID NO: 6):MKRASSGGSRLLAWVLWLQAWRVATPCPGACVCYNEPKVTTSCPQQGLQAVPTGIPASSQRIFLHGNRISHVPAASFQSCRNLTILWLHSNALARIDAAAFTGLTLLEQLDLSDNAQLHVVDPTTFHGLGHLHTLHLDRCGLRELGPGLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFLHGNRIPSVPEHAFRGLHSLDRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLMPLRSLQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCNLPQRLADRDLKRLAASDLEGCAVASGPFRPIQTSQLTDEELLSLPKCCQPDAADKASVLEPGRPASAGNALKGRVPPGDTPPGNGSGPRHINDSPFGTLPSSAEPPLTALRPGGSEPPGLPTTGPRRRPGCSRKNRTRSHCRLGQAGSGASGTGDAEGSGALPALACSLAPLGLALVLWTVLGPC

Full-length Nogo receptor-1 consists of a signal sequence, a N-terminusregion (NT), eight leucine rich repeats (LRR), a LRRCT region (a leucinerich repeat domain C-terminal of the eight leucine rich repeats), aC-terminus region (CT) and a GPI anchor.

The NgR1 domain designations used herein are defined as follows:

TABLE 1 Example NgR1 domains hNgR1 rNgR1 mNgR1 Domain (SEQ ID: 2) (SEQID NO: 4) (SEQ ID NO: 6) Signal Seq.  1-26  1-26  1-26 LRRNT 27-56 27-5627-56 LRR1 57-81 57-81 57-81 LRR2  82-105  82-105  82-105 LRR3 106-130106-130 106-130 LRR4 131-154 131-154 131-154 LRR5 155-178 155-178155-178 LRR6 179-202 179-202 179-202 LRR7 203-226 203-226 203-226 LRR8227-250 227-250 227-250 LRRCT 260-309 260-309 260-309 CTS (CT 310-445310-445 310-445 Signaling) GPI 446-473 446-473 446-473

Soluble Nogo Receptor-1 Polypeptides

Some embodiments of the invention provide a soluble Nogo receptor-1polypeptide. Soluble Nogo receptor-1 polypeptides of the inventioncomprise an NT domain; 8 LRRs and an LRRCT domain and lack a signalsequence and a functional GPI anchor (i.e., no GPI anchor or a GPIanchor that lacks the ability to efficiently associate to a cellmembrane).

In some embodiments, a soluble Nogo receptor-1 polypeptide comprises aheterologous LRR. In some embodiments, a soluble Nogo receptor-1polypeptide comprises 2, 3, 4, 5, 6, 7, or 8 heterologous LRRs. Aheterologous LRR means an LRR obtained from a protein other than Nogoreceptor-1. Exemplary proteins from which a heterologous LRR can beobtained are toll-like receptor (TLR1.2); T-cell activation leucinerepeat rich protein; deceorin; OM-gp; insulin-like growth factor bindingprotein acidic labile subunit slit and robo; and toll-like receptor 4.

In some embodiments, the methods of the invention provide a soluble Nogoreceptor-1 polypeptide of 319 amino acids (soluble Nogo receptor-1 344,sNogoR1-344, or sNogoR344) (residues 26-344 of SEQ ID NOs: 7 and 9 orresidues 27-344 of SEQ ID NO: 9). In some embodiments, the methods ofthe invention provide a soluble Nogo receptor-1 polypeptide of 285 aminoacids (soluble Nogo receptor-1 310, sNogoR1-310, or sNogoR310) (residues26-310 of SEQ ID NOs: 8 and 10 or residues 27-310 of SEQ ID NO: 10).

TABLE 2 Sequences of Human and Rat Nogo Receptor-1 PolypeptidesSEQ ID NO: 7 MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEPK (human 1-344)VTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPAASFRACRNLTILWLHSNVLARIDAAAFTGLALLEQLDLSDNAQLRSVDPATFHGLGRLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNALQALPDDTFRDLGNLTHLFLHGNRISSVPERAFRGLHSLDRLLLHQNRVAHVHPHAFRDLGRLMTLYLFANNLSALPTEALAPLRALQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCSLPQRLAGRDLKRLAANDLQGCAVATGPYHPIWTGRA TDEEPLGLPKCCQPDAADKASEQ ID NO: 8 MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEPK (human 1-310)VTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPAASFRACRNLTILWLHSNVLARIDAAAFTGLALLEQLDLSDNAQLRSVDPATFHGLGRLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNALQALPDDTFRDLGNLTHLFLHGNRISSVPERAFRGLHSLDRLLLHQNRVAHVHPHAFRDLGRLMTLYLFANNLSALPTEALAPLRALQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSE VPCSLPQRLAGRDLKRLAANDLQGCASEQ ID NO: 9 MKRASSGGSRLPTWVLWLQAWRVATPCPGACVCYNEPKV (rat 1-344)TTSRPQQGLQAVPAGIPASSQRIFLHGNRISYVPAASFQSCRNLTILWLHSNALAGIDAAAFTGLTLLEQLDLSDNAQLRVVDPTTFRGLGHLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFLHGNRIPSVPEHAFRGLHSLDRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLVPLRSLQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSGVPSNLPQRLAGRDLKRLATSDLEGCAVASGPFRPFQTNQLT DEELLGLPKCCQPDAADKASEQ ID NO: 10 MKRASSGGSRLPTWVLWLQAWRVATPCPGACVCYNEPKV (rat 1-310)TTSRPQQGLQAVPAGIPASSQRIFLHGNRISYVPAASFQSCRNLTILWLHSNALAGIDAAAFTGLTLLEQLDLSDNAQLRVVDPTTFRGLGHLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFLHGNRIPSVPEHAFRGLHSLDRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLVPLRSLQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSG VPSNLPQRLAGRDLKRLATSDLEGCASEQ ID NO: 11 MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEPK (human 1-310VTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPAASFRAC with alaRNLTILWLHSNVLARIDAAAFTGLALLEQLDLSDNAQLRSV substitutions atDPATFHGLGRLHTLHLDRCGLQELGPGLFRGLAALQYLYLQ amino acidDNALQALPDDTFRDLGNLTHLFLHGNRISSVPERAFRGLHSL positions 266DRLLLHQNRVAHVHPHAFRDLGRLMTLYLFANNLSALPTE and 309)ALAPLRALQYLRLNDNPWVCDARARPLWAWLQKFRGSSSE VPCSLPQRLAGRDLKRLAANDLQGAASEQ ID NO: 12 MKRASSGGSRLPTWVLWLQAWRVATPCPGACVCYNEPKV (rat 1-310 withTTSRPQQGLQAVPAGIPASSQRIFLHGNRISYVPAASFQSCRN ala substitutionsLTILWLHSNALAGIDAAAFTGLTLLEQLDLSDNAQLRVVDP at amino acidTTFRGLGHLHTLHLDRCGLQELGPGLFROLAALQYLYLQD positions 266NNLQALPDNTFRDLGNLTHLFLHGNRLPSVPEHAFRGLHSL and 309)DRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLVPLRSLQYLRLNDNPWVCDARARPLWAWLQKFRGSSSG VPSNLPQRLAGRDLKRLATSDLEGAASEQ ID NO: 13 MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEPK (human 1-344VTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPAASFRAC with alaRNLTILWLHSNVLARIDAAAFTGLALLEQLDLSDNAQLRSV substitutions atDPATFHGLGRLHTLHLDRCGLQELGPGLFRGLAALQYLYLQ amino acidDNALQALPDDTFRDLGNLTHLFLHGNRISSVPERAFRGLHSL positions 266DRLLLHQNRVAHVHPHAFRDLGRLMTLYLFANNLSALPTE and 309)ALAPLRALQYLRLNDNPWVCDARARPLWAWLQKFRGSSSEVPCSLPQRLAGRDLKRLAANDLQGAAVATGPYHPIWTGRA TDEEPLGLPKCCQPDAADKA

In some embodiments of the invention, peripheral administration of asoluble Nogo receptor-1 polypeptide of the invention increases theplasma to brain ratio of Aβ peptide in a mammal or enhances Aβ clearancefrom the brain of a mammal. In some embodiments of the invention,peripheral administration of a soluble Nogo receptor-1 polypeptide ofthe invention improves memory function or inhibits memory loss in amammal. In some embodiments, the mammal is a human.

In some embodiments of the invention, the soluble Nogo receptor-1polypeptide is 70%, 75%, 80%, 85%, 90%, or 95% identical to a referenceamino acid sequence selected from the group consisting of: (i) aminoacids 27 to 310 of SEQ ID NO:2; (ii) amino acids 27 to 344 of SEQ IDNO:2; (iii) amino acids 27 to 445 of SEQ ID NO:2; (iv) amino acids 27 to309 of SEQ ID NO:2; (v) amino acids 1 to 310 of SEQ ID NO:2; (vi) aminoacids 1 to 344 of SEQ ID NO:2; (vii) amino acids 1 to 445 of SEQ IDNO:2; (viii) amino acids 1 to 309 of SEQ ID NO:2; (ix) variants orderivatives of any of said reference amino acid sequences, and (x) acombination of one or more of said reference amino acid sequences orvariants or derivatives thereof.

By “an NgR1 reference amino acid sequence,” or “reference amino acidsequence” is meant the specified sequence without the introduction ofany amino acid substitutions. As one of ordinary skill in the art wouldunderstand, if there are no substitutions, the “isolated polypeptide” ofthe invention comprises an amino acid sequence which is identical to thereference amino acid sequence.

In some embodiments of the invention, the soluble Nogo receptor-1polypeptide is selected from the group consisting of (i) amino acids ato 284 of SEQ ID NO:2, (ii) 210 to b of SEQ ID NO:2 and (iii) a to b ofSEQ ID NO:2, wherein a is any integer from 200 to 210, and b is anyinteger from 284 to 295.

Soluble NgR1 polypeptides for use in the methods of the presentinvention include but are not limited to amino acids amino acids 27 to310 of SEQ ID NO 2; amino acids 27 to 344 of SEQ ID NO:2, amino acids 27to 445 of SEQ ID NO:2, amino acids 27 to 309 of SEQ ID NO:2, amino acids1 to 310 of SEQ ID NO:2, amino acids 1 to 344 of SEQ ID NO:2, aminoacids 1 to 445 of SEQ ID NO:2; and amino acids 1 to 309 of SEQ ID NO:2.

Any different amino acid may be substituted for an amino acid in thepolypeptides of the invention. Amino acids that may be substituted inhuman NgR1 include but are not limited to those amino acids at positions61; 92; 108; 122; 127; 131; 138; 139; 151; 176; 179; 227; 237; 250; 259;108 and 131; 114 and 117; 127 and 151; 127 and 176; 143 and 144; 189 and191; 196 and 199; 202 and 205 256 and 259; 267 and 269; 277 and 279;114, 117 and 139; 189, 191 and 237; 189, 191, and 284; 202, 205 and 227;202, 205 and 250; 220, 223 and 224; 237, 256 and 259; 296, 297 and 300;171, 172, 175 and 176; 292, 296, 297 and 300; 196, 199, 220, 223 and224; 171, 172, 175, 176, 196 and 199; 196, 199, 220, 223, 224 and 250;108, 131 and 61; 36 and 38; 36, 38 and 61; 61, 131, 36 and 38; and 63and 65. Which different amino acid is used depends on a number ofcriteria, for example, the effect of the substitution on theconformation of the polypeptide fragment, the charge of the polypeptidefragment, or the hydrophilicity of the polypeptide fragment. Amino acidsubstitutions for the amino acids of the polypeptides of the inventionand the reference amino acid sequence can include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polars side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Typical amino acids to substitute for amino acids in thereference amino acid sequence include alanine, serine, threonine, inparticular, alanine. Making such substitutions through engineering of apolynucleotide encoding the polypeptide fragment is well within theroutine expertise of one of ordinary skill in the art.

A soluble NgR1 polypeptide can comprise a fragment of at least six,e.g., ten, fifteen, twenty, twenty-five, thirty, forty, fifty, sixty,seventy, one hundred, or more amino acids of SEQ ID NO:2. Correspondingfragments of soluble NgR1 polypeptides at least 70%, 75%, 80%, 85%, 90%,or 95% identical to a reference NgR1 polypeptide of SEQ ID NO:2described herein are also contemplated.

As known in the art, “sequence identity” between two polypeptides isdetermined by comparing the amino acid sequence of one polypeptide tothe sequence of a second polypeptide. When discussed herein, whether anyparticular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95%identical to another polypeptide can be determined using methods andcomputer programs/software known in the art such as, but not limited to,the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981),to find the best segment of homology between two sequences. When usingBESTFIT or any other sequence alignment program to determine whether aparticular sequence is, for example, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference polypeptide sequence and that gaps in homologyof up to 5% of the total number of amino acids in the reference sequenceare allowed.

As discussed below in more detail, soluble NgR1 polypeptides for use inthe methods of the present invention may include any combination of twoor more soluble NgR1 polypeptides. Accordingly, soluble NgR1 polypeptidedimers, either homodimers or heterodimers, are contemplated. Two or moresoluble NgR1 polypeptides as described herein may be directly connected,or may be connected via a suitable peptide linker. Such peptide linkersare described elsewhere herein.

NgR1 polypeptides for use in the methods disclosed herein can becomposed of amino acids joined to each other by peptide bonds ormodified peptide bonds, i.e., peptide isosteres, and may contain aminoacids other than the 20 gene-encoded amino acids (e.g., non-naturallyoccurring amino acids). NgR1 polypeptides, may be modified by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in the NgR1 polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini, or on moieties such as carbohydrates. It will be appreciatedthat the same type of modification may be present in the same or varyingdegrees at several sites in a given NgR1 polypeptide. Also, a given NgR1polypeptide may contain many types of modifications. NgR1 polypeptidesmay be branched, for example, as a result of ubiquitination, and theymay be cyclic, with or without branching. Cyclic, branched, and branchedcyclic NgR1 polypeptides may result from posttranslational naturalprocesses or may be made by synthetic methods. Modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, Proteins—StructureAnd Molecular Properties, T. E. Creighton, W. H. Freeman and Company,New York 2nd Ed., (1993); Posttranslational Covalent Modification OfProteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12(1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al.,Ann IVY Acad Sci 663:48-62 (1992)).

Polypeptides described herein may be cyclic. Cyclization of thepolypeptides reduces the conformational freedom of linear peptides andresults in a more structurally constrained molecule. Many methods ofpeptide cyclization are known in the art. For example, “backbone tobackbone” cyclization by the formation of an amide bond between theN-terminal and the C-terminal amino acid residues of the peptide. The“backbone to backbone” cyclization method includes the formation ofdisulfide bridges between two α-thio amino acid residues (e.g.,cysteine, homocysteine). Certain peptides of the present inventioninclude modifications on the N- and C-terminus of the peptide to form acyclic polypeptide. Such modifications include, but are not limited, tocysteine residues, acetylated cysteine residues, cysteine residues witha NH2 moiety and biotin. Other methods of peptide cyclization aredescribed in Li & Roller, Curr. Top. Med. Chem. 3:325-341 (2002) andU.S. Patent Publication No. U.S. 2005-0260626 A1, which are incorporatedby reference herein in their, entirety.

In methods of the present invention, an NgR1 polypeptide or polypeptidefragment of the invention can be administered directly as a preformedpolypeptide, or indirectly through a nucleic acid vector. In someembodiments of the invention, an NgR1 polypeptide or polypeptidefragment of the invention is administered in a treatment method thatincludes: (1) transforming or transfecting an implantable host cell witha nucleic acid, e.g., a vector, that expresses an NgR1 polypeptide orpolypeptide fragment of the invention; and (2) implanting thetransformed host cell into a mammal, at the site of a disease, disorderor injury. In some embodiments of the invention, the implantable hostcell is removed from a mammal, temporarily cultured, transformed ortransfected with an isolated nucleic acid encoding an NgR1 polypeptideor polypeptide fragment of the invention, and implanted back into thesame mammal from which it was removed. The cell can be, but is notrequired to be, removed from the same site at which it is implanted.Such embodiments, sometimes known as ex vivo gene therapy, can provide acontinuous supply of the NgR1 polypeptide or polypeptide fragment of theinvention, localized at the site of action, for a limited period oftime.

Additional exemplary NgR polypeptides of the invention and methods andmaterials for obtaining these molecules for practicing the presentinvention are described below.

Fusion Proteins and Conjugated Polypeptides

Some embodiments of the invention involve the use of an NgR1 polypeptidethat is not the full-length NgR1 protein, e.g., polypeptide fragments ofNgR1, fused to a heterologous polypeptide moiety to form a fusionprotein. Such fusion proteins can be used to accomplish variousobjectives, e.g., increased serum half-life, improved bioavailability,in vivo targeting to a specific organ or tissue type, improvedrecombinant expression efficiency, improved host cell secretion, ease ofpurification, and higher avidity. Depending on the objective(s) to beachieved, the heterologous moiety can be inert or biologically active.Also, it can be chosen to be stably fused to the NgR1 polypeptide moietyof the invention or to be cleavable, in vitro or in vivo. Heterologousmoieties to accomplish these other objectives are known in the art.

In some embodiments, the soluble Nogo receptor-1 polypeptide of theinvention is a component of a fusion protein that further comprises aheterologous polypeptide. In some embodiments, the heterologouspolypeptide is an immunoglobulin constant domain. In some embodiments,the immunoglobulin constant domain is a heavy chain constant domain. Insome embodiments, the heterologous polypeptide is an Fc fragment. Insome embodiments the Fc is joined to the C-terminal end of the solubleNogo receptor-1 polypeptide of the invention. In some embodiments thefusion Nogo receptor-1 protein is a dimer. The invention furtherencompasses variants, analogs, or derivatives of polypeptide fragmentsas described above.

In some embodiments of the invention, an NgR1 polypeptide fragment canbe fused to one or more additional NgR polypeptide fragments, e.g., anNgR1, NgR2 or NgR3 polypeptide fragment along with Fc.

The human NgR2 polypeptide is shown below as SEQ ID NO:14.

Full-Length Human NgR2 (SEQ ID NO: 14):MLPGLRRLLQ GPASACLLLT LLALPSVTPS CPMLCTCYSSPPTVSCQANN FSSVPLSLPP STQRLFLQNN LIRSLRPGTFGPNLLTLWLF SNNLSTIHPG TFRHLQALEE LDLGDNRHLRSLEPDTFQGL ERLQSLHLYR CQLSSLPGNI FRGLVSLQYLYLQENSLLHL QDDLFADLAN LSHLFLHGNR LRLLTEHVFRGLGSLDRLLL HGNRLQGVHR AAFHGLSRLT ILYLFNNSLASLPGEALADL PALEFLRLNA NPWACDCRAR PLWAWFQRARVSSSDVTCAT PPERQGRDLR ALRDSDFQAC PPPTPTRPGSRARGNSSSNH LYGVAEAGAP PADPSTLYRD LPAEDSRGRQGGDAPTEDDY WGGYGGEDQR GEQTCPGAAC QAPADSRGPA LSAGLRTPLL CLLPLALHHL

The mouse NgR2 polypeptide is shown below as SEQ ID NO:15.

Full-Length Mouse NgR2 (SEQ ID NO: 15):MLPGLRRLLQ GPASACLLLT LLALPSVTPS CPMLCTCYSSPPTVSCQANN FSSVPLSLPP STQRLFLQNN LIRSLRPGTFGPNLLTLWLF SNNLSTIHPG TFRHLQALEE LDLGDNRHLRSLEPDTFQGL ERLQSLHLYR CQLSSLPGNI FRGLVSLQYLYLQENSLLHL QDDLFADLAN LSHLFLHGNR LRLLTEHVFRGLGSLDRLLL HGNRLQGVHR AAFHGLSRLT LLYLFNNSLASLPGEALADL PALEFLRLNA NPWACDCRAR PLWAWFQRARVSSSDVTCAT PPERQGRDLR ALRDSDFQAC PPPTPTRPGSRARGNSSSNH LYGVAEAGAP PADPSTLYRD LPAEDSRGRQGGDAPTEDDY WGGYGGEDQR GEQTCPGAAC QAPADSRGPA LSAGLRTPLL CLLPLALHHL

The human NgR3 polypeptide is shown below as SEQ ID NO:16.

Full-Length Human NgR3 (SEQ ID NO: 16):MLRKGCCVEL LLLLVAAELP LGGGCPRDCV CYPAPMTVSCQAHNFAAIPE GIPVDSERVF LQNNRIGLLQ PGHFSPAMVTLWIYSNNITY IHPSTFEGFV HLEELDLGDN RQLRTLAPETFQGLVKLHAL YLYKCGLSAL PAGVFGGLHS LQYLYLQDNHIEYLQDDIFV DLVNLSHLFL HGNKLWSLGP GTFRGLVNLDRLLLHENQLQ WVHHKAFHDL RRLTTLFLFN NSLSELQGECLAPLGALEFL RLNGNPWDCG CRARSLWEWL QRFRGSSSAVPCVSPGLRHG QDLKLLRAED FRNCTGPASP HQIKSHTLTTTDRAARKEHH SPHGPTRSKG HPHGPRPGHR KPGKNCTNPRNRNQISKAGA GKQAPELPDY APDYQHKFSF DIMPTARPKRKGKCARRTPI RAPSGVQQAS SASSLGASLL AWTLGLAVTL R

The mouse NgR3 polypeptide is shown below as SEQ ID NO:17.

Full-Length Mouse NgR3 (SEQ ID NO: 17):MLRKGCCVEL LLLLLAGELP LGGGCPRDCV CYPAPMTVSCQAHNFAAIPE GIPEDSERIF LQNNRITFLQ QGHFSPAMVTLWIYSNNITF IAPNTFEGFV HLEELDLGDN RQLRTLAPETFQGLVKLHAL YLYKCGLSAL PAGIEGGLHS LQYLYLQDNHLEYLQDDIFV DLVNLSHLFL HGNKLWSLGQ GIFRGLVNLDRLLLHENQLQ WVHHKAFHDL HRLTTLFLFN NSLTELQGDCLAPLVALEFL RLNGNAWDCG CRARSLWEWL RRFRGSSSAVPCATPELRQG QDLKLLRVED FRNCTGPVSP HQIKSHTLTTSDRAARKEHH PSHGASRDKG HPHGHPPGSR SGYKKAGKNCTSHRNRNQIS KVSSGKELTE LQDYAPDYQH KFSFDIMPTARPKRKGKCAR RTPIRAPSGV QQASSGTALG APLLAWILGL AVTLR

In some embodiments of the methods of the invention, the soluble Nogoreceptor polypeptide comprises a first polypeptide fragment and a secondpolypeptide fragment, wherein said first polypeptide fragment comprisesan amino acid sequence identical to a first reference amino acidsequence, except for up to twenty individual amino acid substitutions,wherein said first reference amino acid sequence is selected from thegroup consisting of (a) amino acids a to 445 of SEQ ID NO:2, (b) aminoacids 27 to b of SEQ ID NO:2, and (c) amino acids, a to b of SEQ IDNO:2, wherein a is any integer from 25 to 35, and b is any integer from300 to 450; and wherein said second polypeptide fragment comprises anamino acid sequence identical to a second reference amino acid sequence,except for up to twenty individual amino acid substitutions, whereinsaid second reference amino acid sequence is selected from the groupconsisting of (a) amino acids c to 445 of SEQ ID NO:2, (b) amino acids27 to d of SEQ ID NO:2, and (c) amino acids c to d of SEQ ID NO:2,wherein c is any integer from 25 to 35, and d is any integer from 300 to450.

As an alternative to expression of an NgR fusion polypeptide, a chosenheterologous moiety can be preformed and chemically conjugated to thepolypeptide. In most cases, a chosen heterologous moiety will functionsimilarly, whether fused or conjugated to the NgR1 polypeptide.Therefore, in the following discussion of heterologous amino acidsequences, unless otherwise noted, it is to be understood that theheterologous sequence can be joined to the NgR polypeptide in the formof a fusion protein or as a chemical conjugate.

NgR polypeptides for use in the treatment methods disclosed hereininclude derivatives that are modified, i.e., by the covalent attachmentof any type of molecule such that covalent attachment does not preventthe NgR polypeptide from performing its required function. For example,but not by way of limitation, the NgR polypeptides of the presentinvention may be Modified e.g., by glycosylation, acetylation,pegylation, phosphylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups; proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

The heterologous polypeptide to which the NgR polypeptide is fused isuseful therapeutically or is useful to target the NgR polypeptide. NgRfusion proteins can be used to accomplish various objectives, e.g.,increased serum half-life, improved bioavailability, in vivo targetingto a specific organ or tissue type, improved recombinant expressionefficiency, improved host cell secretion, ease of purification, andhigher avidity. Depending on the objective(s) to be achieved, theheterologous moiety can be inert or biologically active. Also, it can bechosen to be stably fused to the NgR polypeptide or to be cleavable, invitro or in vivo. Heterologous moieties to accomplish these otherobjectives are known in the art.

Pharmacologically active polypeptides such as NgR polypeptides for usein the methods of the present invention may exhibit rapid in vivoclearance, necessitating large doses to achieve therapeuticallyeffective concentrations in the body. In addition, polypeptides smallerthan about 60 kDa potentially undergo glomerular filtration, whichsometimes leads to nephrotoxicity. Fusion or conjugation of relativelysmall polypeptides such as polypeptide fragments of the NgR signalingdomain can be employed to reduce or avoid the risk of suchnephrotoxicity. Various heterologous amino acid sequences, i.e.,polypeptide moieties or “carriers,” for increasing the in vivostability, i.e., serum half-life, of therapeutic polypeptides are known.Examples include serum albumins such as, e.g., bovine serum albumin(BSA) or human serum albumin (HSA).

Due to its long half-life, wide in vivo distribution, and lack ofenzymatic or immunological function, essentially full-length human serumalbumin (HSA), or an HSA fragment, is commonly used as a heterologousmoiety. Through application of methods and materials such as thosetaught in Yeh et al., Proc. Natl. Acad. Sci. USA, 89:1904-08 (1992) andSyed et al., Blood 89:3243-52 (1997), HSA can be used to form a fusionprotein or polypeptide conjugate that displays pharmacological activityby virtue of the NgR polypeptide moiety while displaying significantlyincreased in vivo stability, e.g., 10-fold to 100-fold higher. TheC-terminus of the HSA can be fused to the N-terminus of the NgRpolypeptide moiety. Since HSA is a naturally secreted protein, the HSAsignal sequence can be exploited to obtain secretion of the fusionprotein into the cell culture medium when the fusion protein is producedin a eukaryotic, e.g., mammalian, expression system.

In certain embodiments, NgR polypeptides for use in the methods of thepresent invention further comprise a targeting moiety. Targetingmoieties include a protein or a peptide which directs localization to acertain part of the body.

Some embodiments of the invention employ an NgR polypeptide moiety fusedto a hinge and Fc region, i.e., the C-terminal portion of an Ig heavychain constant region. In some embodiments, amino acids in the hingeregion may be substituted with different amino acids. Exemplary aminoacid substitutions for the hinge region according to these embodimentsinclude substitutions of individual cysteine residues in the hingeregion with different amino acids. Any different amino acid may besubstituted for a cysteine in the hinge region. Amino acid substitutionsfor the amino acids of the polypeptides of the invention and thereference amino acid sequence can include amino acids with basic sidechains (e.g.; lysine, arginine, histidine), acidic side chains (e.g.;aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Typical aminoacids to substitute for cysteines in the reference amino acid includealanine, serine, threonine, in particular, serine and alanine. Makingsuch substitutions through engineering of a polynucleotide encoding thepolypeptide fragment is well within the routine expertise of one ofordinary skill in the art.

Potential advantages of an NgR-polypeptide-Fc fusion include solubility,in vivo stability, and multivalency, e.g., dimerization. The Fc regionused can be an IgA, IgD, or IgG Fc region (hinge-CH2-CH3).Alternatively, it can be an IgE or IgM Fc region (hinge-CH2-CH3-CH4). AnIgG Fc region is generally used, e.g., an IgG1 Fc region or IgG4 Fcregion. Materials and Methods for constructing and expressing DNAencoding Fc fusions are known in the art and can be applied to obtainfusions without undue experimentation. Some embodiments of the inventionemploy a fusion protein such as those described in Capon et al., U.S.Pat. Nos. 5,428,130 and 5,565,335.

The signal sequence is a polynucleotide that encodes an amino acidsequence that initiates transport of a protein across the membrane ofthe endoplasmic reticulum. Signal sequences useful for constructing animmunofusin include antibody light chain signal sequences, e.g.,antibody 14.18 (Gillies et al., J. Immunol. Meth., 125:191-202. (1989)),antibody heavy chain signal sequences, e.g., the MOPC141 antibody heavychain signal: sequence (Sakano et al., Nature 286:5774 (1980)).Alternatively, other signal sequences can be used. See, e.g., Watson,Nucl. Acids Res. 12:5145 (1984). The signal peptide is usually cleavedin the lumen of the endoplasmic reticulum by signal peptidases. Thisresults in the secretion of an immunofusin protein containing the Fcregion and the NgR1 polypeptide moiety.

In some embodiments, the DNA sequence may encode a proteolytic cleavagesite between the secretion cassette and the NgR1 polypeptide moiety.Such a cleavage site may provide, e.g., for the proteolytic cleavage ofthe encoded fusion protein, thus separating the Fc domain from thetarget protein. Useful proteolytic cleavage sites include amino acidsequences recognized by proteolytic enzymes such as trypsin, plasmin,thrombin, factor Xa, or enterokinase K.

The secretion cassette can be incorporated into a replicable expressionvector. Useful vectors include linear nucleic acids, plasmids,phagemids, cosmids and the like. An exemplary expression vector is pdC,in which the transcription of the immunofusin DNA is placed under thecontrol of the enhancer and promoter of the human cytomegalovirus. See,e.g., Lo et al., Biochim. Biophys. Acta 1088:712 (1991); and Lo et al.,Protein Engineering 11:495-500 (1998). An appropriate host cell can betransformed or transfected with a DNA that encodes an NgR1 polypeptideor polypeptide fragment of the invention and used for the expression andsecretion of the polypeptide. Host cells that are typically used includeimmortal hybridoma cells, myeloma cells, 293 cells, Chinese hamsterovary (CHO) cells, Hela cells, and COS cells.

Fully intact, wild-type Fc regions display effector functions thatnormally are unnecessary and undesired in an Fc fusion protein used inthe methods of the present invention. Therefore, certain binding sitestypically are deleted from the Fe region during the construction of thesecretion cassette. For example, since coexpression with the light chainis unnecessary, the binding site for the heavy chain binding protein,Bip (Hendershot et al., Immunol. Today 8:111-14 (1987)), is deleted fromthe CH2 domain of the Fc region of IgE, such that this site does notinterfere with the efficient secretion of the immunofusin. Transmembranedomain sequences, such as those present in IgM, also are generallydeleted.

The IgG1 Fc region is most often used. Alternatively, the Fc region ofthe other subclasses of immunoglobulin gamma (gamma-2, gamma-3 andgamma-4) can be used in the secretion cassette. The IgG1 Fc region ofimmunoglobulin gamma-1 is generally used in the secretion cassette andincludes at least part of the hinge region, the CH2 region, and the CH3region. In some embodiments, the Fc region of immunoglobulin gamma-1 isa CH2-deleted-Fc, which includes part of the hinge region and the CH3region, but not the CH2 region. A CH2-deleted-Fe has been described byGillies et al., Hum. Antibod. Hybridomas 1:47 (1990). In someembodiments, the Fc region of one of IgA, IgD, IgE, or IgM, is used.

NgR-polypeptide-moiety-Fc fusion proteins can be constructed in severaldifferent configurations. In one configuration, the C-terminus of theNgR polypeptide moiety is fused directly to the N-terminus of the Fchinge moiety. In a slightly different configuration, a shortpolypeptide, e.g., 2-10 amino acids, is incorporated into the fusionbetween the N-terminus of the NgR polypeptide moiety and the C-terminusof the Fc moiety. In the alternative configuration, the shortpolypeptide is incorporated into the fusion between the C-terminus ofthe NgR polypeptide moiety and the N-terminus of the Fc moiety. Anexemplary embodiment of this configuration is NgR1(310)-2XG4S-Fc, whichis amino acids 26-310 of SEQ ID NO:2 linked to (Gly-Gly-Gly-Gly-Ser)₂(SEQ ID NO:19) which is linked to Fc. Such a linker providesconformational flexibility, which may improve biological activity insome circumstances. If a sufficient portion of the hinge region isretained in the Fc moiety, the NgR-polypeptide-moiety-Fc fusion willdimerize, thus forming a divalent molecule. A homogeneous population ofmonomeric Fc fusions will yield monospecific, bivalent dimers. A mixtureof two monomeric Fc fusions each having a different specificity willyield bispecific, bivalent dimers.

Any of a number of cross-linkers that contain a correspondingamino-reactive group and thiol-reactive group can be used to link an NgRpolypeptide or polypeptide fragment of the invention to serum albumin.Examples of suitable linkers include amine reactive cross-linkers thatinsert a thiol-reactive maleimide, e.g., SMCC, AMAS, BMPS, MBS, EMCS,SMPB, SMPH, KMUS, and GMBS. Other suitable linkers insert athiol-reactive haloacetate group, e.g., SBAP, SIA, SIAB. Linkers thatprovide a protected or non-protected thiol for reaction with sulfhydrylgroups to product a reducible linkage include SPDP, SMPT, SATA, andSATP. Such reagents are commercially available (e.g., Pierce ChemicalCompany, Rockford, Ill.).

Conjugation does not have to involve the N-terminus of an NgRpolypeptide or polypeptide fragment of the invention or the thiol moietyon serum albumin. For example, NgR-polypeptide-albumin fusions can beobtained using genetic engineering techniques, wherein the NgRpolypeptide moiety is fused to the serum albumin gene at its.N-terminus, C-terminus, or both.

NgR polypeptides of the invention can be fused to a polypeptide tag. Theterm “polypeptide tag,” as used herein, is intended to mean any sequenceof amino acids that can be attached to, connected to, or linked to anNgR polypeptide and that can be used to identify, purify, concentrate orisolate the NgR polypeptide. The attachment of the polypeptide tag tothe NgR polypeptide may occur, e.g., by constructing a nucleic acidmolecule that comprises: (a) a nucleic acid sequence that encodes thepolypeptide tag, and (b) a nucleic acid sequence that encodes an NgRpolypeptide. Exemplary polypeptide tags include, e.g., amino acidsequences that are capable of being post-translationally modified, e.g.,amino acid sequences that are biotinylated. Other exemplary polypeptidetags include, e.g., amino acid sequences that are capable of beingrecognized and/or bound by an antibody (or fragment thereof) or otherspecific binding reagent. Polypeptide tags that are capable of beingrecognized by an antibody (or fragment thereof) or other specificbinding reagent include, e.g., those that are known in the art as“epitope tags.” An epitope tag may be a natural or an artificial epitopetag. Natural and artificial epitope tags are known in the art,including, e.g., artificial epitopes such as FLAG, Strep, orpoly-histidine peptides. FLAG peptides include the sequenceAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:27) orAsp-Tyr-Lys-Asp-Glu-Asp-Asp-Lys (SEQ ID NO:28) (Einhauer, A. andJungbauer, A., J. Biochem. Biophys. Methods 49:1-3:455-465 (2001)). TheStrep epitope has the sequence Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQID NO:29). The VSV-G epitope can also be used and has the sequenceTyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys (SEQ ID NO:30). Anotherartificial epitope is a poly-His sequence having six histidine residues(His-His-His-His-His-His (SEQ ID NO:31). Naturally-occurring epitopesinclude the influenza virus hemagglutinin (HA) sequenceTyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ile-Glu-Gly-Arg (SEQ ID NO:32)recognized by the monoclonal antibody 12CA5 (Murray et al., Anal.Biochem. 229:170-179 (1995)) and the eleven amino acid sequence fromhuman c-myc (Myc) recognized by the monoclonal antibody 9E10(Glu-Gln-Lys-Leu-Leu-Ser-Glu-Glu-Asp-Leu-Asn (SEQ ID NO:33) (Manstein etal., Gene 162:129-134 (1995)). Another useful epitope is the tripeptideGlu-Glu-Phe which is recognized by the monoclonal antibody YL 1/2.(Stammers et al. FEBS Lett. 283:298-302 (1991)).

In certain embodiments, the NgR polypeptide and the polypeptide tag maybe connected via a linking amino acid sequence. As used herein, a“linking amino acid sequence” may be an amino acid sequence that iscapable of being recognized and/or cleaved by one or more proteases.Amino acid sequences that can be recognized and/or cleaved by one ormore proteases are known in the art. Exemplary amino acid sequences arethose that are recognized by the following proteases: factor VIIa,factor IXa, factor Xa, APC, t-PA, u-PA, trypsin, chymotrypsin,enterokinase, pepsin, cathepsin B, H, L, S, D, cathepsin G, renin,angiotensin converting enzyme, matrix metalloproteases (collagenases,stromelysins, gelatinases), macrophage elastase, Cir, and Cis. The aminoacid sequences that are recognized by the aforementioned proteases areknown in the art. Exemplary sequences recognized by certain proteasescan be found, e.g., in U.S. Pat. No. 5,811,252.

Polypeptide tags can facilitate purification using commerciallyavailable chromatography media.

In some embodiments of the invention, an NgR polypeptide fusionconstruct is used to enhance the production of an NgR polypeptide moietyin bacteria. In such constructs, a bacterial protein normally expressedand/or secreted at a high level is employed as the N-terminal fusionpartner of an NgR1 polypeptide or polypeptide fragment of the invention.See, e.g., Smith et al., Gene 67:31 (1988); Hopp et al., Biotechnology6:1204 (1988); La Vallie et al., Biotechnology 11:187 (1993).

By fusing an NgR polypeptide moiety at the amino and carboxy termini ofa suitable fusion partner, bivalent or tetravalent forms of an NgRpolypeptide or polypeptide fragment of the invention can be obtained.For example, an NgR polypeptide moiety can be fused to the amino andcarboxy termini of an Ig moiety to produce a bivalent monomericpolypeptide containing two NgR polypeptide moieties. Upon dimerizationof two of these monomers, by virtue of the Ig moiety, a tetravalent formof an NgR-polypeptide is obtained. Such multivalent forms can be used toachieve increased binding affinity for the target. Multivalent forms ofan NgR polypeptide or polypeptide fragment of the invention also can beobtained by placing NgR polypeptide moieties in tandem to formconcatamers, which can be employed alone or fused to a fusion partnersuch as Ig or HSA.

Conjugated Polymers (Other than Polypeptides)

Some embodiments of the invention involve an NgR polypeptide orpolypeptide fragment of the invention wherein one or more polymers areconjugated (covalently linked) to the NgR polypeptide. Examples ofpolymers suitable for such conjugation include polypeptides (discussedabove), sugar polymers and polyalkylene glycol chains. Typically, butnot necessarily, a polymer is conjugated to the NgR polypeptide orpolypeptide fragment of the invention for the purpose of improving oneor more of the following: Solubility, stability, or bioavailability.

The class of polymer generally used for conjugation to an NgRpolypeptide or polypeptide fragment of the invention is a polyalkyleneglycol. Polyethylene glycol (PEG) is most frequently used. PEG moieties,e.g., 1, 2, 3, 4 or 5 PEG polymers; can be conjugated to each NgRpolypeptide to increase serum half life, as compared to the NgRpolypeptide alone. PEG moieties are non-antigenic and essentiallybiologically inert. PEG moieties used in the practice of the inventionmay be branched or unbranched.

The number of PEG moieties attached to the NgR polypeptide and themolecular weight of the individual PEG chains can vary. In general, thehigher the molecular weight of the polymer, the fewer polymer chainsattached to the polypeptide. Usually; the total polymer mass attached toan NgR polypeptide or polypeptide fragment is from 20 kDa to 40 kDa.Thus, if one polymer chain is attached, the molecular weight of thechain is generally 20-40 kDa. If two chains are attached, the molecularweight of each chain is generally 10-20 kDa. If three chains areattached, the molecular weight is generally 7-14 kDa.

The polymer, e.g., PEG, can be linked to the NgR polypeptide through anysuitable, exposed reactive group on the polypeptide. The exposedreactive group(s) can be, e.g., an N-terminal amino group or the epsilonamino group of an internal lysine residue, or both. An activated polymercan react and covalently link at any free amino group on the NgRpolypeptide. Free carboxylic groups, suitably activated carbonyl groups,hydroxyl, guanidyl, imidazole, oxidized carbohydrate moieties andmercapto groups of the NgR polypeptide (if available) also can be usedas reactive groups for polymer attachment.

In a conjugation reaction, from about 1.0 to about 10 moles of activatedpolymer per mole of polypeptide, depending on polypeptide concentration,is typically employed. Usually, the ratio chosen represents a balancebetween maximizing the reaction while minimizing side reactions (oftennon-specific) that can impair the desired pharmacological activity ofthe NgR polypeptide moiety. Preferably, at least 50% of the biologicalactivity (as demonstrated, e.g., in any of the assays described hereinor known in the art) of the NgR polypeptide is retained, and mostpreferably nearly 100% is retained.

The polymer can be conjugated to the NgR polypeptide using conventionalchemistry. For example, a polyalkylene glycol moiety can be coupled to alysine epsilon amino group of the NgR polypeptide. Linkage to the lysineside chain can be performed with an N-hydroxylsuccinimide (NHS) activeester such as PEG succinimidyl succinate (SS-PEG) and succinimidylpropionate (SPA-PEG). Suitable polyalkylene glycol moieties include,e.g., carboxymethyl-NHS and norleucine-NHS, SC. These reagents arecommercially available. Additional amine-reactive PEG linkers can besubstituted for the succinimidyl moiety. These include, e.g.,isothiocyanates, nitrophenylcarbonates (PNP), epoxides, benzotriazolecarbonates, SC-PEG, tresylate, aldehyde, epoxide, carbonylimidazole andPNP carbonate. Conditions are usually optimized to maximize theselectivity and extent of reaction. Such optimization of reactionconditions is within ordinary skill in the art.

PEGylation can be carried out by any of the PEGylation reactions knownin the art. See, e.g., Focus on Growth Factors, 3: 4-10, 1992 andEuropean patent applications EP 0 154 316 and EP 0 401 384. PEGylationmay be carried out using an acylation reaction or an alkylation reactionwith a reactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer).

PEGylation by acylation generally involves reacting an active esterderivative of polyethylene glycol. Any reactive PEG molecule can beemployed in the PEGylation. PEG esterified to N-hydroxysuccinimide (NHS)is a frequently used activated PEG ester. As used herein, “acylation”includes without limitation the following types of linkages between thetherapeutic protein and a water-soluble polymer such as PEG: amide,carbamate, urethane, and the like. See, e.g., Bioconjugate Chem. 5:133-140, 1994. Reaction parameters are generally selected to avoidtemperature, solvent, and pH conditions that would damage or inactivatethe NgR polypeptide.

Generally, the connecting linkage is an amide and typically at least 95%of the resulting product is mono-, di- or tri-PEGylated. However, somespecies with higher degrees of PEGylation may be formed in amountsdepending on the specific reaction conditions used. Optionally, purifiedPEGylated species are separated from the mixture, particularly unreactedspecies, by conventional purification methods, including, e.g.,dialysis; salting-out, ultrafiltration, ion-exchange chromatography, gelfiltration chromatography, hydrophobic exchange chromatography; andelectrophoresis.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with an NgR1 polypeptide or polypeptide fragment ofthe invention in the presence of a reducing agent. In addition, one canmanipulate the reaction conditions to favor PEGylation substantiallyonly at the N-terminal amino group of the NgR polypeptide, i.e. amono-PEGylated protein. In either case of mono-PEGylation orpoly-PEGylation, the PEG groups are typically attached to the proteinvia a —CH2-NH— group. With particular reference to the —CH2- group, thistype of linkage is known as an “alkyl” linkage.

Derivatization via reductive alkylation to produce an N-terminallytargeted mono-PEGylated product exploits differential reactivity ofdifferent types of primary amino groups (lysine versus the N-terminal)available for derivatization. The reaction is performed at a pH thatallows one to take advantage of the pKa differences between theepsilon-amino groups of the lysine residues and that of the N-terminalamino group of the protein. By such selective derivatization, attachmentof a water-soluble polymer that contains a reactive group, such as analdehyde, to a protein is controlled: the conjugation with the polymertakes place predominantly at the N-terminus of the protein and nosignificant modification of other reactive groups, such as the lysineside chain amino groups, occurs.

The polymer molecules used in both the acylation and alkylationapproaches are selected from among water-soluble polymers. The polymerselected is typically modified to have a single reactive group, such asan active ester for acylation or an aldehyde for alkylation, so that thedegree of polymerization may be controlled as provided for in thepresent methods. An exemplary reactive PEG aldehyde is polyethyleneglycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy oraryloxy derivatives thereof (see, e.g., Harris et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. For the acylationreactions, the polymer(s) selected typically have a single reactiveester group. For reductive alkylation, the polymer(s) selected typicallyhave a single reactive aldehyde group. Generally, the water-solublepolymer will not be selected from naturally occurring glycosyl residues,because these are usually made more conveniently by mammalianrecombinant expression systems.

Methods for preparing a PEGylated NgR polypeptides of the inventiongenerally includes the steps of (a) reacting an NgR1 polypeptide orpolypeptide fragment of the invention with polyethylene glycol (such asa reactive ester or aldehyde derivative of PEG) under conditions wherebythe molecule becomes attached to one or more PEG groups, and (b)obtaining the reaction product(s). In general, the optimal reactionconditions for the acylation reactions will be determined case-by-casebased on known parameters and the desired result. For example, a largerthe ratio of PEG to protein, generally leads to a greater the percentageof poly-PEGylated product.

Reductive alkylation to produce a substantially homogeneous populationof mono-polymer/NgR polypeptide generally includes the steps of (a)reacting an NgR1 polypeptide or polypeptide fragment of the inventionwith a reactive PEG molecule under reductive alkylation conditions, at apH suitable to permit selective modification of the N-terminal aminogroup of NgR; and (b) obtaining the reaction product(s).

For a substantially homogeneous population of mono-polymer/NgRpolypeptide, the reductive alkylation reaction conditions are those thatpermit the selective attachment of the water-soluble polymer moiety tothe N-terminus of a NgR polypeptide or polypeptide fragment of theinvention. Such reaction conditions generally provide for pKadifferences between the lysine side chain amino groups and theN-terminal amino group. For purposes of the present invention, the pH isgenerally in the range of 3-9, typically 3-6.

NgR polypeptides of the invention can include a tag, e.g., a moiety thatcan be subsequently released by proteolysis. Thus, the lysine moiety canbe selectively modified by first reacting a His-tag modified with alow-molecular-weight linker such as Traut's reagent (Pierce ChemicalCompany, Rockford, Ill.) which will react with both the lysine andN-terminus, and then releasing the His tag. The polypeptide will thencontain a free SH group that can be selectively modified with a PEGcontaining a thiol-reactive head group such as a maleimide group, avinylsulfone group, a haloacetate group, or a free or protected SH.

Traut's reagent can be replaced with any linker that will set up aspecific site for PEG attachment. For example, Traut's reagent can bereplaced with SPDP, SMPT, SATA, or SATP (Pierce Chemical Company,Rockford, Ill.). Similarly one could react the protein with anamine-reactive linker that inserts a maleimide (for example SMCC, AMAS,BMPS, MBS, EMCS, SMPB, SMPH, KMUS, or GMBS), a haloacetate group (SBAP,SIA, SIAB), or a vinylsulfone group and react the resulting product witha PEG that contains a free SH.

In some embodiments, the polyalkylene glycol moiety is coupled to acysteine group of the NgR polypeptide. Coupling can be effected using,e.g., a maleimide group, a vinylsulfone group, a haloacetate group, or athiol group.

Optionally, the NgR polypeptide is conjugated to the polyethylene-glycolmoiety through a labile bond. The labile bond can be cleaved in, e.g.,biochemical hydrolysis, proteolysis, or sulfhydryl cleavage. Forexample, the bond can be cleaved under in vivo (physiological)conditions.

The reactions may take place by any suitable method used for reactingbiologically active materials with inert polymers, generally at about pH5-8, e.g., pH 5, 6, 7, or 8, if the reactive groups are on the alphaamino group at the N-terminus. Generally the process involves preparingan activated polymer and thereafter reacting the protein with theactivated polymer to produce the soluble protein suitable forformulation.

Nucleic Acid Molecules of the Present Invention

The human Nogo receptor-1 polynucleotide is shown below as SEQ ID NO:1.

Full-Length Human Nogo receptor-1 is encoded by nucleotide 166 to nucleotide1587 of SEQ ID NO: 1:agcccagcca gagccgggcg gagcggagcg cgccgagcct cgtcccgcggccgggccggg gccgggccgt agcggcggcg cctggatgcg gacccggccgcggggagacg ggcgcccgcc ccgaaacgac tttcagtccc cgacgcgccccgcccaaccc ctacgatgaa gagggcgtcc gctggaggga gccggctgctggcatgggtg ctgtggctgc aggcctggca ggtggcagcc ccatgcccaggtgcctgcgt atgctacaat gagcccaagg tgacgacaag ctgcccccagcagggcctgc aggctgtgcc cgtgggcatc cctgctgcca gccagcgcatcttcctgcac ggcaaccgca tctcgcatgt gccagctgcc agcttccgtgcctgccgcaa cctcaccatc ctgtggctgc.actcgaatgt gctggcccgaattgatgcgg ctgccttcac tggcctggcc ctcctggagc agctggacctcagcgataat gcacagctcc ggtctgtgga ccctgccaca ttccacggcctgggccgcct acacacgctg cacctggacc gctgcggcct gcaggagctgggcccggggc tgttccgcgg cctggctgcc ctgcagtacc tctacctgcaggacaacgcg ctgcaggcac tgcctgatga caccttccgc gacctgggcaacctcacaca cctcttcctg cacggcaacc gcatctccag cgtgcccgagcgcgccttcc gtgggctgca cagcctcgac cgtctcctac tgcaccagaaccgcgtggcc catgtgcacc cgcatgcctt ccgtgacctt ggccgcctcatgacactcta tctgtttgcc aacaatctat cagcgctgcc cactgaggccctggcccccc tgcgtgccct gcagtacctg aggctcaacg acaacccctgggtgtgtgac tgccgggcac gcccactctg ggcctggctg cagaagttccgcggctcctc ctccgaggtg ccctgcagcc tcccgcaacg cctggctggccgtgacctca aacgcctagc tgccaatgac ctgcagggct gcgctgtggccaccggccct taccatccca tctggaccgg cagggccacc gatgaggagccgctggggct tcccaagtgc tgccagccag atgccgctga caaggcctcagtactggagc ctggaagacc agcttcggca ggcaatgcgc tgaagggacgcgtgccgccc ggtgacagcc cgccgggcaa cggctctggc ccacggcacatcaatgactc accctttggg actctgcctg gctctgctga gcccccgctcactgcagtgc ggcccgaggg ctccgagcca ccagggttcc ccacctcgggccctcgccgg aggccaggct gtccacgcaa gaaccgcacc cgcagccactgccgtctggg ccaggcaggc agcgggggtg gcgggactgg tgactcagaaggctcaggtg ccctacccag cctcacctgc agcctcaccc ccctgggcctggcgctggtg ctgtggacag tgcttgggcc ctgctgaccc ccagcggacacaagagcgtg ctcagcagcc aggtgtgtgt acatacgggg tctctctccacgccgccaag ccagccgggc ggccgacccg tggggcaggc caggccaggtcctccctgat ggacgcctg

The rat Nogo receptor-1 polynucleotide is shown below as SEQ ID NO:3.

atgaagaggg cgtcctccgg aggaagccgg ctgccgacat gggtgttatggctacaggcc tggagggtag caacgccctg ccctggtgcc tgtgtgtgctacaatgagcc caaggtcaca acaagccgcc cccagcaggg cctgcaggctgtacccgctg gcatcccagc ctccagccag agaatcttcc tgcacggcaaccgaatctct tacgtgccag ccgccagctt ccagtcatgc cggaatctcaccatcctgtg gctgcactca aatgcgctgg ccgggattga tgccgcggccttcactggtc tgaccctcct ggagcaacta gatcttagtg acaatgcacagctccgtgtc gtggacccca ccacgttccg tggcctgggc cacctgcacacgctgcacct agaccgatgc ggcctgcagg agctggggcc tggcctattccgtgggctgg cagctctgca gtacctctac ctacaagaca acaacctgcaggcacttccc gacaacacct tccgagacct gggcaacctc acgcatctctttctgcatgg caaccgtatc cccagtgttc ctgagcacgc tttccgtggcttgcacagtc ttgaccgtct cctcttgcac cagaaccatg tggctcgtgtgcacccacat gccttccggg accttggccg actcatgacc ctctacctgtttgccaacaa cctctccatg ctccccgcag aggtcctagt gcccctgaggtctctgcagt acctgcgact caatgacaac ccctgggtgt gtgactgcagggcacgtccg ctctgggcct ggctgcagaa gttccgaggt tcctcatccggggtgcccag caacctaccc caacgcctgg caggccgtga tctgaagcgcctggctacca gtgacttaga gggttgtgct gtggcttcgg ggcccttccgtcccttccag accaatcagc tcactgatga ggagctgctg ggcctccccaagtgctgcca gccggatgct gcagacaagg cctcagtact ggaacccgggaggccggcgt ctgttggaaa tgcactcaag ggacgtgtgc ctcccggtgacactccacca ggcaatggct caggcccacg gcacatcaat gactctccatttgggacttt gcccggctct gcagagcccc cactgactgc cctgcggcctgggggttccg agcccccggg actgcccacc acgggccccc gcaggaggccaggttgttcc agaaagaacc gcacccgtag ccactgccgt ctgggccaggcaggaagtgg gagcagtgga actggggatg cagaaggttc gggggccctgcctgccctgg cctgcagcct tgctcctctg ggccttgcac tggtactttggacagtgctt gggccctgct ga

The mouse Nogo receptor-1 polynucleotide is shown below as SEQ ID NO:5.

Full-Length Mouse Nogo receptor-1 is encoded by nucleotide 178 to nucleotide1599 of SEQ ID NO: 5:agccgcagcc cgcgagccca gcccggcccg gtagagcgga gcgccggagcctcgtcccgc ggccgggccg ggaccgggcc ggagcagcgg cgcctggatgcggacccggc cgcgcgcaga cgggcgcccg ccccgaagcc gcttccagtgcccgacgcgc cccgctcgac cccgaagatg aagagggcgt cctccggaggaagcaggctg ctggcatggg tgttatggct acaggcctgg agggtagcaacaccatgccc tggtgcttgt gtgtgctaca atgagcccaa ggtaacaacaagctgccccc agcagggtct gcaggctgtg cccactggca tcccagcctctagccagcga atcttcctgc atggcaaccg aatctctcac gtgccagctgcgagcttcca gtcatgccga aatctcacta tcctgtggct gcactctaatgcgctggctc ggatcgatgc tgctgccttc actggtctga ccctcctggagcaactagat cttagtgata atgcacagct tcatgtcgtg gaccctaccacgttccacgg cctgggccac ctgcacacac tgcacctaga ccgatgtggcctgcgggagc tgggtcccgg cctattccgt ggactagcag ctctgcagtacctctaccta caagacaaca atctgcaggc actccctgac aacacctttcgagacctggg caacctcacg catctctttc tgcatggcaa ccgtatccccagtgtgcctg agcacgcttt ccgtggcctg cacagtcttg accgcctcctcttgcaccag aaccatgtgg ctcgtgtgca cccacatgcc ttccgggaccttggccgcct catgaccctc tacctgtttg ccaacaacct ctccatgctgcctgcagagg tcctaatgcc cctgaggtct ctgcagtacc tgcgactcaatgacaacccc tgggtgtgtg actgccgggc acgtccactc tgggcctggctgcagaagtt ccgaggttcc tcatcagagg tgccctgcaa cctgccccaacgcctggcag accgtgatct taagcgcctc gctgccagtg acctagagggctgtgctgtg gcttcaggac ccttccgtcc catccagacc agtcagctcactgatgagga gctgctgagc ctccccaagt gctgccagcc agatgctgcagacaaagcct cagtactgga acccgggagg ccagcttctg ccggaaacgccctcaaggga cgtgtgcctc ccggtgacac tccaccaggc aatggctcaggccctcggca catcaatgac tctccatttg gaactttgcc cagctctgcagagccdccac tgactgccct gcggcctggg ggttccgagc caccaggacttcccaccact ggtccccgca ggaggccagg ttgttcccgg aagaatcgcacccgcagcca ctgccgtctg ggccaggcgg gaagtggggc cagtggaacaggggacgcag agggttcagg ggctctgcct gctctggcct gcagccttgctcctctgggc cttgcactgg tactttggac agtgcttggg ccctgctgaccagccaccag ccaccaggtg tgtgtacata tggggtetcd ctccacgccgccagccagag ccagggacag gctctgaggg gcaggccaSg ccctccctgacagatgcctc cccaccagcc cacccccatc tccaccccat catgtttacagggttccggg ggtggcgttt gttccagaac gccacctccc acccggatcgcggtatatag agatatgaat tttattttac ttgtgtaaaa tatcggatgacgtggaataa agagctcttt tcttaaaaaa aaaaaaaaaa aa

The present invention provide a polynucleotide that encodes any of therecited polypeptides or polypeptide fragments of the invention.

In some embodiments, the nucleic acid encodes a polypeptide selectedfrom the group consisting of amino acid residues 26-344 of Nogoreceptor-1 as shown in SEQ ID NOs: 7 and 9 or amino acid residues 27-344of Nogo receptor-1 as shown in SEQ ID NO: 9. In some embodiments, thenucleic acid molecule encodes a polypeptide §elected from the groupconsisting of amino acid residues 26-310 of Nogo receptor-1 as shown inSEQ ID NOs: 8 and 10 or amino acid residues 27-310 of Nogo receptor-1 asshown in SEQ ID NO: 10. As used herein, “nucleic acid” means genomicDNA, cDNA, mRNA and antisense molecules, as well as nucleic acids basedon alternative backbones or including alternative bases whether derivedfrom natural sources or synthesized. In some embodiments, the nucleicacid further comprises a transcriptional promoter and optionally asignal sequence each of which is operably linked to the nucleotidesequence encoding the polypeptides of the invention.

In some embodiments, the invention provides a nucleic acid encoding aNogo receptor-1 fusion protein of the invention, including a fusionprotein comprising a polypeptide selected from the group consisting ofamino acid residues 26-344 of Nogo receptor-1 as shown in SEQ ID NOs: 7and 9 or amino acid residues 27-344 of SEQ ID NO: 9 and amino acidresidues 26-310 of Nogo receptor-1 as shown in SEQ ID NOs: 8 and 10 oramino acid residues 27-310 of SEQ ID NO: 10. In some embodiments, thenucleic acid encoding a Nogo receptor-1 fusion protein further comprisesa transcriptional promoter and optionally a signal sequence. In someembodiments, the nucleotide sequence further encodes an immunoglobulinconstant region. In some embodiments, the immunoglobulin constant regionis a heavy chain constant region. In some embodiments, the nucleotidesequence further encodes an immunoglobulin heavy chain constant regionjoined to a hinge region. In some embodiments the nucleic acid furtherencodes Fc. In some embodiments the Nogo receptor-1 fusion proteinscomprise an Fc fragment.

The encoding nucleic acids of the present invention may further bemodified so as to contain a detectable label for diagnostic and probepurposes. A variety of such labels are known in the art and can readilybe employed with the encoding molecules herein described. Suitablelabels include, but are not limited to, biotin, radiolabeled nucleotidesand the like. A skilled artisan can employ any of the art known labelsto obtain a labeled encoding nucleic acid molecule.

The present invention also includes polynucleotides that hybridize undermoderately stringent or high stringency conditions to the noncodingstrand, or complement, of a polynucleotide that encodes any one of thepolypeptides of the invention. In some embodiments, polynucleotides thathybridize encode a polypeptide of the invention. Stringent conditionsare known to those skilled in the art and can be found in CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6.

Compositions

In some embodiments, the invention provides compositions comprising asoluble Nogo receptor polypeptide or fusion protein of the presentinvention.

In some embodiments, the invention provides a composition comprising apolynucleotide of the present invention.

In some embodiments, the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compound's intopreparations which can be used pharmaceutically for delivery to the siteof action. Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol and dextran.Optionally, the suspension may also contain stabilizers. Liposomes canalso be used to encapsulate the molecules of this invention for deliveryinto the cell. Exemplary “pharmaceutically acceptable carriers” are anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible, water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In some embodiments, the composition comprisesisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride. In some embodiments, the compositionscomprise pharmaceutically acceptable substances such as wetting or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the soluble Nogo receptors or fusion proteins of the invention.

Compositions of the invention may be in a variety of forms, including,for example, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions. The preferred form depends on the intended mode ofadministration and therapeutic application. In one embodiment,compositions are in the form of injectable or infusible solutions, suchas compositions similar to those used for passive immunization of humanswith other antibodies.

The composition can be formulated as a solution, micro emulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In some embodiments, the active compound may be prepared with a carrierthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York (1978).

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a polypeptide(s), or fusion protein of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the solubleNogo receptor polypeptide or Nogo receptor fusion protein may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual. A therapeutically effective amount is also one in whichany toxic or detrimental effects of the soluble Nogo receptorpolypeptide or Nogo receptor fusion protein are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage unit formas used herein refers to physically discrete units suited as unitarydosages for the mammalian subjects to be treated, each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the soluble receptor polypeptide or Nogo receptorfusion protein and the particular therapeutic or prophylactic effect tobe achieved, and (b) the limitations inherent in the art of compoundingsuch a soluble receptor polypeptide or Nogo receptor fusion protein forthe treatment of sensitivity in individuals. In some embodiments atherapeutically effective dose range for the soluble Nogo receptorpolypeptide 0.001-10 mg/Kg per day. In some embodiments atherapeutically effective dose range for soluble Nogo receptorpolypeptides thereof is 0.01-1 mg/Kg per day. In some embodiments atherapeutically effective dose range for the Nogo receptor polypeptidesthereof is 0.05-0.5 mg/Kg per day.

For treatment with a soluble NgR1 receptor polypeptide of the invention,the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and moreusually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For exampledosages can be 1 mg/kg body weight or 10 mg/kg body weight or within therange of 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate inthe above ranges are also intended to be within the scope of theinvention. Subjects can be administered such doses daily, on alternativedays, weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly.

In some methods, two or more soluble NgR1 receptor polypeptides orfusion proteins are administered simultaneously, in which case thedosage of each polypeptide or fusion protein administered falls withinthe ranges indicated. Supplementary active compounds also can beincorporated into the compositions used in the methods of the invention.For example, a NgR polypeptide or fusion protein may be coformulatedwith and/or coadministered with one or more additional therapeuticagents, such as an adrenergic; anti-adrenergic, anti-androgen,anti-anginal, anti-anxiety, anticonvulsant, antidepressant,anti-epileptic, antihyperlipidemic, antihyperlipoproteinemic,antihypertensive, anti-inflammatory, antiobessional, antiparkinsonian,antipsychotic, adrenocortical steroid; adrenocortical suppressant;aldosterone antagonist; amino acid; anabolic steroid; analeptic;androgen; blood glucose regulator; cardioprotectant; cardiovascular;cholinergic agonist or antagonist; cholinesterase deactivator orinhibitor, cognition adjuvant or enhancer; dopaminergic; enzymeinhibitor, estrogen, free oxygen radical scavenger; GABA agonist;glutamate antagonist; hormone; hypocholesterolemic; hypolipidemic;hypotensive; immunizing; immunostimulant; monoamine oxidase inhibitor,neuroprotective; NMDA antagonist; AMPA antagonist, competitive or-non-competitive NMDA antagonist; opioid antagonist; potassium channelopener; non-hormonal sterol derivative; post-stroke and post-head traumatreatment; prostaglandin; psychotropic; relaxant; sedative;sedative-hypnotic; selective adenosine antagonist; serotonin antagonist;serotonin inhibitor; selective serotonin uptake inhibitor; serotoninreceptor antagonist; sodium and calcium channel blocker; steroid;stimulant; and thyroid hormone and inhibitor agents.

In embodiments of the present invention, the NgR polypeptide or fusionprotein is delivered peripheral to the central nervous system.“Peripheral to the central nervous system” includes any route ofadministration except for those routes of administration wherein the NgRpolypeptide is administered directly to the central nervous system,e.g., intracerebroventricularly, or intrathecally.

In some embodiments, the NgR polypeptide or fusion protein isadministered by a route selected from the group consisting of oraladministration; nasal administration; parenteral administration;transdermal administration; topical administration; intraocularadministration; intrabronchial administration; intraperitonealadministration; intravenous administration; subcutaneous administration;intramuscular administration; and a combination of two or more of theseroutes of administration. In one embodiment, the NgR polypeptide orfusion protein is administered subcutaneously.

Parenteral injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Additionally, oneapproach for parenteral administration employs the implantation of aslow-release or sustained-released systems, which assures that aconstant level of dosage is maintained, according to U.S. Pat. No.3,710,795, incorporated herein by reference in its entirety.

The invention encompasses any suitable delivery method for a NgRpolypeptide or fusion protein to a selected target tissue, includingbolus injection of an aqueous solution or implantation of acontrolled-release system. Use of a controlled-release implant reducesthe need for repeat injections.

The compositions may also comprise a NgR polypeptide or fusion proteinof the invention dispersed in a biocompatible carrier material thatfunctions as a suitable delivery or support system for the compounds.Suitable examples of sustained release carriers include semipermeablepolymer matrices in the form of shaped articles such as suppositories orcapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers ofL-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers22:547-56 (1985)); poly(2-hydroxyethyl-methacrylate), ethylene vinylacetate (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981);Langer, Chem. Tech. 12:98-105 (1982)) or poly-D-(−)-3hydroxybutyric acid(EP 133,988).

Vectors of the Invention

Vectors comprising nucleic acids encoding the soluble NgR polypeptidesmay be used to produce soluble polypeptides for use in the methods ofthe invention. The choice of vector and expression control sequences towhich such nucleic acids are operably linked depends on the functionalproperties desired, e.g., protein expression, and the host cell to betransformed.

In a typical embodiment, a soluble NgR polypeptide useful in the methodsdescribed herein is a recombinant protein produced by a cell (e.g., aCHO cell) that carries an exogenous nucleic acid encoding the protein.In other embodiments, the recombinant polypeptide is produced by aprocess commonly known as gene activation, wherein a cell that carriesan exogenous nucleic acid that includes a promoter or enhancer isoperably linked to an endogenous nucleic acid that encodes thepolypeptide.

Routine techniques for making recombinant polypeptides (e.g.,recombinant soluble NgR polypeptides) may be used to constructexpression vectors encoding the polypeptides of interest usingappropriate transcriptional/translational control signals and theprotein coding sequences. (See, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, 3d Ed. (Cold Spring Harbor Laboratory2001)). These methods may include in vitro recombinant DNA and synthetictechniques and in vivo recombination, e.g., in vivo homologousrecombination. Expression of a nucleic acid sequence encoding apolypeptide may be regulated by a second nucleic acid sequence that isoperably linked to the polypeptide encoding sequence such that thepolypeptide is expressed in a host transformed with the recombinant DNAmolecule.

Expression control elements useful for regulating the expression of anoperably linked coding sequence are known in the art. Examples include,but are not limited to, inducible promoters, constitutive promoters,secretion signals, and other regulatory elements. When an induciblepromoter is used, it can be controlled, e.g., by a change in nutrientstatus, or a change in temperature, in the host cell medium.

Expression vectors capable of being replicated in a bacterial oreukaryotic host comprising a nucleic acid encoding a polypeptide areused to transfect a host and thereby direct expression of such nucleicacid to produce the polypeptide, which may then be isolated. Thepreferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Routine techniques for transfecting cells withexogenous DNA sequences may be used in the present invention.Transfection methods may include chemical means, e.g.; calciumphosphate, DEAE-dextran, or liposome; or physical means, e.g.,microinjection or electroporation. The transfected cells are grown up byroutine techniques. For examples, see Kuchler et al. (1977) BiochemicalMethods in Cell Culture and Virology. The expression products areisolated from the cell medium in those systems where the protein issecreted from the host cell, or from the cell suspension afterdisruption of the host cell system by, e.g., routine mechanical,chemical, or enzymatic means. These methods may also be carried outusing cells that have been genetically modified by other procedures,including gene targeting and gene activation (see Treco et al. WO95/31560, herein incorporated by reference; see also Selden et al. WO93/09222).

The vector can include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule extra-chromosomally in a bacterial hostcell. Such replicons are well known in the art. In addition, vectorsthat include a prokaryotic replicon may also include a gene whoseexpression confers a detectable marker such as a drug resistance.Examples of bacterial drug-resistance genes are those that conferresistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can also include aprokaryotic or bacteriophage promoter for directing expression of thecoding gene sequences in a bacterial host cell. Promoter sequencescompatible with bacterial hosts are typically provided in plasmidvectors containing convenient restriction sites for insertion of a DNAsegment to be expressed. Examples of such plasmid vectors are pUC8,pUC9, pBR322 and pBR329 (BioRad), pPL and pKK223 (Pharmacia). Anysuitable prokaryotic host can be used to express a recombinant DNAmolecule encoding a protein used in the methods of the invention.

For the purposes of this invention, numerous expression vector systemsmay be employed. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, adeno-associated virus, herpes simplexvirus-1, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV)or SV40 virus. Examples of such vectors can be found in PCT publicationsWO 2006/060089 and WO2002/056918 which are incorporated herein in theirentirties. Others involve the use of polycistronic systems with internalribosome binding sites. Additionally, cells which have integrated theDNA into their chromosomes may be selected by introducing one or moremarkers which allow selection of transfected host cells. The marker mayprovide for prototrophy to an auxotrophic host, biocide resistance(e.g., antibiotics) or resistance to heavy metals such as copper. Theselectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell bycotransformation. The neomycin phosphotransferase (neo) gene is anexample of a selectable marker gene (Southern et al., J. Mol. Anal.Genet. 1:327-341 (1982)). Additional elements may also be needed foroptimal synthesis of mRNA. These elements may include signal sequences,splice signals, as well as transcriptional promoters, enhancers, andtermination signals.

In one embodiment, a proprietary expression vector of Biogen IDEC, Inc.,referred to as NEOSPLA (U.S. Pat. No. 6,159,730) may be used. Thisvector contains the cytomegalovirus promoter/enhancer, the mouse betaglobin major promoter, the SV40 origin of replication, the bovine growthhormone polyadenylation sequence, neomycin phosphotransferase exon 1 andexon 2, the dihydrofolate reductase gene and leader sequence. Thisvector has been found to result in very high level expression upontransfection in CHO cells, followed by selection in G418 containingmedium and methotrexate amplification. Of course, any expression vectorwhich is capable of eliciting expression in eukaryotic cells may be usedin the present invention. Examples of suitable vectors include, but arenot limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS,pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2(available from Invitrogen, San Diego, Calif.), and plasmid pCI(available from Promega, Madison, Wis.). Additional eukaryotic cellexpression vectors are known in the art and are commercially available.Typically, such vectors contain convenient restriction sites forinsertion of the desired DNA segment. Exemplary vectors include pSVL andpKSV-10 (Pharmacia), pBPV-1, pm12d (International Biotechnologies),pTDT1 (ATCC 31255), retroviral expression vector pMIG and pLL3.7,adenovirus shuttle vector pDC315, and AAV vectors. Other exemplaryvector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.

In general, screening large numbers of transformed cells for those whichexpress suitably high levels of the antagonist is routineexperimentation which can be carried out, for example, by roboticsystems.

The recombinant expression vectors may carry sequences that regulatereplication of the vector in host cells (e.g., origins of replication)and selectable marker genes. It will be appreciated by those skilled inthe art that the design of the expression vector, including theselection of regulatory sequences may depend on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, etc. Frequently used regulatory sequences for mammalianhost cell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and enhancersderived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMVpromoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdmlP)), polyoma and strong mammalian promoters such as nativeimmunoglobulin and actin promoters. For further description of viralregulatory elements, and sequences thereof, see e.g., Stinski, U.S. Pat.No. 5,168,062; Bell, U.S. Pat. No. 4,510,245; and Schaffner, U.S. Pat.No. 4,968,615.

The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., Axel, U.S. Pat. Nos.4,399,216; 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to a drug, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Frequently used selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr− host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

Vectors comprising polynucleotides encoding soluble NgR polypeptides canbe used for transformation of a suitable host cell. Transformation canbe by any suitable method. Methods for introduction of exogenous DNAinto mammalian cells are well known in the art and includedextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei. In addition, nucleic acidmolecules may be introduced into mammalian cells by viral vectors.

Transformation of host cells can be accomplished by conventional methodssuited to the vector and host cell employed. For transformation ofprokaryotic host cells, electroporation and salt treatment methods canbe employed (Cohen et al., Proc. Natl. Acad. Sci. USA 69:2110-14(1972)). For transformation of vertebrate cells, electroporation,cationic lipid or salt treatment methods can be employed. See, e.g.,Graham et al., Virology 52:456-467 (1973); Wigler et al., Proc. Natl.Acad. Sci. USA 76:1373-76 (1979).

The host cell line used for protein expression is most preferably ofmammalian origin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to NSO, SP2 cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells DG44 andDUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervicalcarcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mousefibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma),P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells),RAH (human lymphocyte) and 293 (human kidney). Host cell lines aretypically available from commercial services, the American TissueCulture Collection or from published literature.

Expression of polypeptides from production cell lines can be enhancedusing known techniques. For example, the glutamine synthetase (GS)system is commonly used for enhancing expression under certainconditions. See, e.g., European Patent Nos. 0 216 846, 0 256 055, and 0323 997 and European Patent Application No. 89303964.4.

In some embodiments, the invention provides recombinant DNA molecules(rDNA) that contain a coding sequence. As used herein, a rDNA moleculeis a DNA molecule that has been subjected to molecular manipulation.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989). In some rDNA molecules, acoding DNA sequence is operably linked to expression control sequencesand vector sequences. A vector of the present invention may be at leastcapable of directing the replication or insertion into the hostchromosome, and preferably also expression, of the structural geneincluded in the rDNA molecule.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form a rDNAmolecules that contains a coding sequence. Eukaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. Typically, such vectors are provided containingconvenient restriction sites for insertion of the desired DNA segment.Examples of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1, pML2d(International Biotechnologies), pTDT1 (ATCC® 31255) and othereukaryotic expression vectors.

Eukaryotic cell expression vectors used to construct the rDNA moleculesof the present invention may further include a selectable marker that iseffective in an eukaryotic cell, preferably a drug resistance selectionmarker. A preferred drug resistance marker is the gene whose expressionresults in neomycin resistance, i.e., the neomycin phosphotransferase(neo) gene. (Southern et al., J. Mol. Anal. Genet. 1:327-341 (1982)).Alternatively, the selectable marker can be present on a separateplasmid, the two vectors introduced by co-transfection of the host cell,and transfectants selected by culturing in the appropriate drug for theselectable marker.

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains are inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. Expression vectorsinclude plasmids, retroviruses, cosmids, YACs, EBV-derived episomes, andthe like. The antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors. In some embodiments, bothgenes are inserted into the same expression vector. The antibody genesare inserted into the expression vector by standard methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites arepresent).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can be easilyinserted and expressed, as described above. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C region, and also atthe splice regions that occur within the human C_(H) exons.Polyadenylation and transcription termination occur at nativechromosomal sites downstream of the coding regions. The recombinantexpression vector can also encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The antibody chaingene may be cloned into the vector such that the signal peptide islinked in-frame to the amino terminus of the antibody chain gene. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

Other embodiments of the invention use a lentiviral vector forexpression of the polynucleotides of the invention. Lentiviruses caninfect noncycling and postmitotic cells, and also provide the advantageof not being silenced during development allowing generation oftransgenic animals through infection of embryonic stem cells. Milhavetet al., Pharmacological Rev. 55:629-648 (2003). Other polynucleotideexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus.

Transcription of the polynucleotides of the invention can be driven froma promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II(pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters are expressed at high levels in all cells; the levelsof a given pol II promoter in a given cell type depends on the nature ofthe gene regulatory sequences (enhancers, silencers, etc.) presentnearby. Prokaryotic RNA polymerase promoters are also used, providingthat the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells (Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA87:6743-7 (1990); Gao and Huang, Nucleic Acids Res. 21:2867-72 (1993);Lieber et al., Methods Enzymol. 217:47-66 (1993); Zhou et al., Mol.Cell. Biol. 10:4529-37 (1990)). Several investigators have demonstratedthat polynucleotides expressed from such promoters can function inmammalian cells (e.g. Kashani-Sabet et al., Antisense Res. Dev. 2:3-15(1992); Ojwang et al., Proc. Natl. Acad. Sci. USA 89:10802-6 (1992);Chen et al., Nucleic Acids Res. 20:4581-9 (1992); Yu et al., Proc. Natl.Acad. Sci. USA 90:6340-4 (1993); L'Huillier et al., EMBO J. 11:4411-8(1992); Lisziewicz et al., Proc. Natl. Acad. Sci. U.S.A 90:8000-4(1993); Thompson et al., Nucleic Acids Res. 23:2259 (1995); Sullenger &Cech, Science 262:1566 (1993)).

Host Cells and Methods of Recombinantly Producing Protein of theInvention

Nucleic acid molecules encoding soluble Nogo receptor polypeptides,soluble Nogo receptor fusion proteins of this invention and vectorscomprising these nucleic acid molecules can be used for transformationof a suitable host cell. Transformation can be by any known method forintroducing polynucleotides into a host cell. Methods for introductionof heterologous polynucleotides into mammalian cells are well known inthe art and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.

Transformation of appropriate cell hosts with a rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed. With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods can be employed (see, for example, Sambrook et al.,Molecular Cloning—A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989); Cohen et al., Proc. Natl. Acad. Sci. USA 69:2110-2114(1972)). With regard to transformation of vertebrate cells with vectorscontaining rDNA, electroporation, cationic lipid or salt treatmentmethods can be employed (see, for example, Graham et al., Virology.52:456-467 (1973); Wigler et al., Proc. Natl. Acad. Sci. USA76:1373-1376 (1979)).

Successfully transformed cells, i.e., cells that contain a rDNA moleculeof the present invention, can be identified by well known techniquesincluding the selection for a selectable marker. For example, cellsresulting from the introduction of an rDNA of the present invention canbe cloned to produce single colonies. Cells from those colonies can beharvested, lysed and their DNA content examined for the presence of therDNA using a method such as that described by Southern, J. Mol. Biol.98:503-517 (1975) or the proteins produced from the cell may be assayedby an immunological method.

Host cells for expression of a polypeptide or antibody of the inventionfor use in a method of the invention may be prokaryotic or eukaryotic.Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC®). These include, inter alia,Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, babyhamster kidney (MK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a numberof other cell lines. Cell lines of particular preference are selectedthrough determining which cell lines have high expression levels. Otheruseful eukaryotic host cells include plant cells. Other cell lines thatmay be used are insect cell lines, such as Sf9 cells. Exemplaryprokaryotic host cells are E. coli and Streptomyces.

When recombinant expression vectors encoding the soluble Nogo receptorpolypeptides and soluble Nogo receptor fusion proteins of the inventionare introduced into mammalian host cells, they are produced by culturingthe host cells for a period of time sufficient to allow for expressionof the antibody, polypeptide and fusion polypeptide in the host cellsor, more preferably, secretion of the soluble Nogo receptor polypeptidesand soluble Nogo receptor fusion proteins of the invention into theculture medium in which the host cells are grown. Soluble Nogo receptorpolypeptides and soluble Nogo receptor fusion proteins of the inventioncan be recovered from the culture medium using standard proteinpurification methods.

Further, expression of soluble Nogo receptor polypeptides and solubleNogo receptor fusion proteins of the invention of the invention (orother moieties therefrom) from production cell lines can be enhancedusing a number of known techniques. For example, the glutaminesynthetase gene expression system (the GS system) is a common approachfor enhancing expression under certain conditions. The GS system isdiscussed in whole or part in connection with European Patent Nos. 0 216846, 0 256 055, and 0 323 997 and European Patent Application No.89303964.4.

A polypeptide produced by a cultured cell as described herein can berecovered from the culture medium as a secreted polypeptide, or, if itis not secreted by the cells, it can be recovered from host celllysates. As a first step in isolating the polypeptide, the culturemedium or lysate is generally centrifuged to remove particulate celldebris. The polypeptide thereafter is isolated, and preferably purified,from contaminating soluble proteins and other cellular components, withthe following procedures being exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS PAGE;ammonium sulfate precipitation; and gel filtration, e.g., with Sephadex™columns (Amersham Biosciences). Protease inhibitors may be used toinhibit proteolytic degradation during purification. One skilled in theart will appreciate that purification methods suitable for thepolypeptide of interest may require modification to account for changesin the character of the polypeptide upon expression in recombinant cellculture.

The purification of polypeptides may require the use of, e.g., affinitychromatography, conventional ion exchange chromatography, sizingchromatography, hydrophobic interaction chromatography, reverse phasechromatography, gel filtration or other conventional proteinpurification techniques. See, e.g., Deutscher, ed. (1990) “Guide toProtein Purification” in Methods in Enzymology, Vol. 182.

Cell Therapy

In some embodiments of the invention a soluble NgR polypeptide isadministered in a treatment method that includes: (1) transforming ortransfecting an implantable host cell with a nucleic acid, e.g., avector, that expresses a soluble NgR polypeptide; and (2) implanting thetransformed host cell into a mammal, at the site of a disease, disorderor injury. For example, the transformed host cell can be implanted atthe site of a spinal cord injury. In some embodiments of the invention,the implantable host cell is removed from a mammal, temporarilycultured, transformed or transfected with an isolated nucleic acidencoding a soluble NgR polypeptide, and implanted back into the samemammal from which it was removed. The cell can be, but is not requiredto be, removed from the same site at which it is implanted. Suchembodiments, sometimes known as ex vivo gene therapy, can provide acontinuous supply of the soluble NgR polypeptide, localized at the siteof site of action, for a limited period of time.

Gene Therapy

A soluble NgR polypeptide can be produced in vivo in a mammal, e.g., ahuman patient, using a gene-therapy approach to treatment of a disease,disorder or injury in which reducing Aβ accumulation would betherapeutically beneficial. This involves administration of a suitablesoluble NgR polypeptide-encoding nucleic acid operably linked tosuitable expression control sequences. Generally, these sequences areincorporated into a viral vector. Suitable viral vectors for such genetherapy include an adenoviral vector, an alphavirus vector, anenterovirus vector, a pestivirus vector, a lentiviral vector, abaculoviral vector, a herpesvirus vector, an Epstein Barr viral vector,a papovaviral vector, a poxvirus vector, a vaccinia viral vector,adeno-associated viral vector and a herpes simplex viral vector. Theviral vector can be a replication-defective viral vector. Adenoviralvectors that have a deletion in its E1 gene or E3 gene are typicallyused. When an adenoviral vector is used, the vector usually does nothave a selectable marker gene. Examples of such vectors can be found inPCT publications WO 2006/060089 and WO2002/056918 which are incorporatedherein in their entirties.

Expression constructs of a soluble NgR polypeptide may be administeredin any biologically effective carrier, e.g. any formulation orcomposition capable of effectively delivering the soluble NgRpolypeptide gene to cells in vivo. Approaches include insertion of thesubject gene in viral vectors including recombinant retroviruses,adenovirus, adeno-associated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly; plasmid DNA can be delivered with the help of, forexample, cationic liposomes (lipofectin) or derivatized (e.g. antibodyconjugated), polylysine conjugates, gramacidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding a soluble NgR polypeptide, or a soluble NgR polypeptideantisense nucleic acid. Infection of cells with a viral vector has theadvantage that a large proportion of the targeted cells can receive thenucleic acid. Additionally, molecules encoded within the viral vector,e.g., by a cDNA contained in the viral vector, are expressed efficientlyin cells which have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous genes invivo, particularly into humans. These vectors provide efficient deliveryof genes into cells, and the transferred nucleic acids are stablyintegrated into the chromosomal DNA of the host. The development ofspecialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes. Areplication defective retrovirus can be packaged into virions which canbe used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are known to those skilled in theart. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include .psi.Crip,.psi.Cre, .psi.2 and .psi.Am. Retroviruses have been used to introduce avariety of genes into many different cell types, including epithelialcells, in vitro and/or in vivo (see for example Eglitis, et al. (1985)Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci.USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

Another viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See, for example, Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 d1324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in theart. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells and can be used to infect a wide variety of cell types, includingepithelial cells (Rosenfeld et al. (1992) cited supra). Furthermore, thevirus particle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situ whereintroduced DNA becomes integrated into the host genome retroviral DNA).Moreover, the carrying capacity of the adenoviral genome for foreign DNAis large (up to 8 kilobases) relative to other gene delivery vectors(Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol.57:267).

Yet another viral vector system useful for delivery of the subject geneis the adeno-associated virus (AAV). Reviewed in A11, 2004, NovartisFound Symp. 255:165-78; and Lu, 2004, Stem Cells Dev. 13(1):133-45.Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (Fora review see Muzyczka et al. (1992) Curr. Topics in Micro. and Immunol.158:97-129). It is also one of the few viruses that may integrate itsDNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing aslittle as 300 base pairs of AAV can be packaged and can integrate. Spacefor exogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a solubleNgR polypeptide, fragment, or analog, in the tissue of an animal. Mostnonviral methods of gene transfer rely on normal mechanisms used bymammalian cells for the uptake and intracellular transport ofmacromolecules. In preferred embodiments, non-viral gene deliverysystems of the present invention rely on endocytic pathways for theuptake of the subject NgR gene by the targeted cell. Exemplary genedelivery systems of this type include liposomal derived systems,poly-lysine conjugates, and artificial viral envelopes. Otherembodiments include plasmid injection systems such as are described inMeuli et al. (2001) J Invest Dermatol. 116(1):131-135; Cohen et al.(2000) Gene Ther 7(22):1896-905; or Tam et al. (2000) Gene Ther7(21):1867-74.

In a representative embodiment, a gene encoding a soluble NgRpolypeptide, active fragment, or analog, can be entrapped in liposomesbearing positive charges on their surface (e.g., lipofectins) and(optionally) which are tagged with antibodies against cell surfaceantigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka20:547-551; PCT publication WO91/06309; Japanese patent application1047381; and European patent publication EP-A-43075).

In clinical settings, the gene delivery systems for the therapeutic NgRgene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g. by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) Pros. Natl. Acad. Sci. USA 91: 3054-3057).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

Guidance regarding gene therapy in particular for treating a CNScondition or disorder as described herein can be found, e.g., in U.S.patent application Ser. No. 2002/0,193,335 (provides methods ofdelivering a gene therapy vector, or transformed cell, to neurologicaltissue); U.S. patent application Ser. No. 2002/0,187,951 (providesmethods for treating a neurodegenerative disease using a lentiviralvector to a target cell in the brain or nervous system of a mammal);U.S. patent application Ser. No. 2002/0,107,213 (discloses a genetherapy vehicle and methods for its use in the treatment and preventionof neurodegenerative disease); U.S. patent application Ser. No.2003/0,099,671 (discloses a mutated rabies virus suitable for deliveringa gene to the CNS); and U.S. Pat. No. 6,436,708 (discloses a genedelivery system which results in long-term expression throughout thebrain); U.S. Pat. No. 6,140,111 (discloses retroviral vectors suitablefor human gene therapy in the treatment of a variety of disease); andKaspar et al. (2002) Mol. Ther. 5:50-6; Suhr et al (1999) Arch Neurol.56:287-92; and Wong et al. (2002) Nat Neurosci 5, 633-639).

Production of Recombinant Proteins Using a rDNA Molecule

The present invention further provides methods for producing a solubleNogo receptor polypeptide and/or soluble Nogo receptor fusion protein ofthe invention using nucleic acid molecules herein described. In generalterms, the production of a recombinant form of a protein typicallyinvolves the following steps. First, a nucleic acid molecule is obtainedthat encodes a protein of the invention. If the encoding sequence isuninterrupted by introns, it is directly suitable for expression in anyhost. The nucleic acid molecule is then optionally placed in operablelinkage with suitable control sequences, as described above, to form anexpression unit containing the protein open reading frame. Theexpression unit is used to transform a suitable host and the transformedhost is cultured under conditions that allow the production of therecombinant protein. Optionally the recombinant protein is isolated fromthe medium or from the cells; recovery and purification of the proteinmay not be necessary in some instances where some impurities may betolerated.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using appropriate replicons and control sequences, as setforth above. The control sequences, expression vectors, andtransformation methods are dependent on the type of host cell used toexpress the gene and were discussed in detail earlier. Suitablerestriction sites can, if not normally available, be added to the endsof the coding sequence so as to provide an excisable gene to insert intothese vectors. A skilled artisan can readily adapt any host/expressionsystem known in the art for use with the nucleic acid molecules of theinvention to produce recombinant protein.

Methods Using Soluble NgR polypeptides, Fusion Proteins, Polynucleotidesand Compositions

One embodiment of the present invention provides a method for increasingthe plasma to brain ratio of Aβ peptide in a mammal, comprisingadministering a therapeutically effective amount of a soluble Nogoreceptor polypeptide peripheral to the central nervous system.

Another embodiment of the invention provides a method for enhancing Aβclearance from the brain of a mammal, comprising administering atherapeutically effective amount of a soluble Nogo receptor polypeptideperipheral to the central nervous system.

A further embodiment of the invention provides a method for improvingmemory function or inhibiting memory loss in a mammal comprisingadministering a therapeutically effective amount of a soluble Nogoreceptor polypeptide peripheral to the central nervous system.

Another embodiment of the invention provides a method of reducing thenumber of Aβ plaques in the brain of a mammal, comprising administeringto a mammal in need thereof a therapeutically effective amount of asoluble Nogo receptor polypeptide, wherein said administration isperipheral to the central nervous system.

Another embodiment of the invention provides a method of reducing thesize of Aβ plaques in the brain of a mammal, comprising administering toa mammal in need thereof a therapeutically effective amount of a solubleNogo receptor polypeptide, wherein said administration is peripheral tothe central nervous system.

Another embodiment of the invention provides a method of treating adisease associated with Aβ plaque accumulation in a mammal comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of a soluble Nogo receptor polypeptide, wherein saidadministration is peripheral to the central nervous system.

Disease that can be treated using the methods of the present inventioninclude but are not limited to Alzheimer's disease, mild cognitiveimpairment, mild-to-moderate cognitive impairment, vascular dementia,cerebral amyloid angiopathy, hereditary cerebral hemorrhage, seniledementia, Down's syndrome, inclusion body myositis, age-related maculardegeneration, primary amyloidosis, secondary amyloidosis or a conditionassociated with Alzheimer's disease. Conditions associated withAlzheimer's disease that can be treated using the methods of the presentinvention include but are not limited to hypothyroidism, cerebrovasculardisease, cardiovascular disease, memory loss, anxiety, a behavioraldysfunction, a neurological condition, or a psychological condition.Behavioral dysfunction that can be treated using the methods of thepresent invention include but is not limited to apathy, aggression, orincontinence. Neurological conditions that can be treated using themethods of the present invention include but are not limited toHuntington's disease, amyotrophic lateral sclerosis, acquiredimmunodeficiency, Parkinson's disease, aphasia, apraxia, agnosia, Pickdisease, dementia with Lewy bodies, altered muscle tone, seizures,sensory loss, visual field deficits, incoordination, gait disturbance,transient ischemic attack or stroke, transient alertness, attentiondeficit, frequent falls, syncope, neuroleptic sensitivity, normalpressure hydrocephalus, subdural hematoma, brain tumor, posttraumaticbrain injury, or posthypoxic damage. Psychological conditions that canbe treated using the methods of the present invention include but arenot limited to depression, delusions, illusions, hallucinations, sexualdisorders, weight loss, psychosis, a sleep disturbance, insomnia,behavioral disinhibition, poor insight, suicidal ideation, depressedmood, irritability, anhedonia, social withdrawal, or excessive guilt.

Mild cognitive impairment (MCI) is a condition characterized by a stateof mild but measurable impairment in thinking skills, but is notnecessarily associated with the presence of dementia. MCI frequently,but not necessarily, precedes Alzheimer's disease. It is a diagnosisthat has most often been associated with mild memory problems, but itcan also be characterized by mild impairments in other thinking skills,such as language or planning skills. However, in general, an individualwith MCI will have more significant memory lapses than would be expectedfor someone of their age or educational background. As the conditionprogresses, a physician may change the diagnosis to Mild-to-ModerateCognition Impairment, as is well understood in this art.

In methods of the present invention, a soluble NgR polypeptide isadministered peripheral to the central nervous system. “Peripheral tothe central nervous system” includes any route of administration exceptfor those routes of administration wherein the NgR polypeptide isadministered directly to the central nervous system, e.g.,intracerebroventricularly, or intrathecally. The soluble Nogo receptorpolypeptides or Nogo receptor fusion proteins of the present inventioncan be administered via parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, inhalational or buccalroutes. For example, an agent may be administered locally to a site ofinjury via microinfusion. In one embodiment, the soluble NgR polypeptideis administered subcutaneously. In some embodiments of the presentinvention, the soluble NgR polypeptide does not cross the blood-brainbarrier (BBB).

The soluble Nogo receptor polypeptides or fusion proteins of the presentinvention can be provided alone, or in combination, or in sequentialcombination with other agents that modulate a particular pathologicalprocess. As used herein, the soluble Nogo receptor and Nogo receptorfusion proteins, are said to be administered in combination with one ormore additional therapeutic agents when the two are administeredsimultaneously, consecutively or independently.

In some embodiments, an NgR receptor polypeptide or fusion protein maybe coformulated with and/or coadministered with one or more anti-Aβantibodies for use in the methods of the present invention. Examples ofanti-Aβ for use in the methods of the present invention can be found,e.g., in U.S. Patent Publication Nos. 20060165682 A1, 20060039906 A1,and 20040043418 A1.

In some embodiments, an NgR1 polypeptide or fusion protein may becoformulated with and/or coadministered with one or more additionaltherapeutic agents, such as an adrenergic agent, anti-adrenergic agent,anti-androgen agent, anti-anginal agent, anti-anxiety agent,anticonvulsant agent, antidepressant agent, anti-epileptic agent,antihyperlipidemic agent, antihyperlipoproteinemic agent,antihypertensive agent, anti-inflammatory agent, antiobessional agent,antiparkinsonian agent, antipsychotic agent, adrenocortical steroid;adrenocortical suppressant; aldosterone antagonist; amino acid; anabolicsteroid; analeptic agent; androgen; blood glucose regulator;cardioprotectant agent; cardiovascular agent; cholinergic agonist orantagonist; cholinesterase deactivator or inhibitor, cognition adjuvantor enhancer; dopaminergic agent; enzyme inhibitor, estrogen, free oxygenradical scavenger; GABA agonist; glutamate antagonist; hormone;hypocholesterolemic agent; hypolipidemic agent; hypotensive agent;immunizing agent; immunostimulant agent; monoamine oxidase inhibitor,neuroprotective agent; NMDA antagonist; AMPA antagonist, competitive or-non-competitive NMDA antagonist; opioid antagonist; potassium channelopener; non-hormonal sterol derivative; post-stroke and post-head traumatreatment; prostaglandin agent; psychotropic agent; relaxant agent;sedative agent; sedative-hypnotic agent; selective adenosine antagonist;serotonin antagonist; serotonin inhibitor; selective serotonin uptakeinhibitor; serotonin receptor antagonist; sodium and calcium channelblocker; steroid; stimulant; and thyroid hormone and inhibitor agentsfor use in the methods of the present invention.

The dosage administered will be dependent upon the age, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. The compounds ofthis invention can be utilized in vivo, ordinarily in mammals, such ashumans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or invitro.

The methods of treatment of diseases and disorders as described hereinare typically tested in vitro, and then in vivo in an acceptable animalmodel, for the desired therapeutic or prophylactic activity, prior touse in humans. Suitable animal models, including transgenic animals, arewill known to those of ordinary skill in the art. The effect of the NgR1polypeptides, fusion proteins, and compositions on increasing the brainto plasma ratio of Aβ peptide and enhancing Aβ clearance from the brainand reducing the number of Aβ plaques can be tested in vitro asdescribed in the Examples. Finally, in vivo tests can be performed bycreating transgenic mice which express the appropriate phenotype andadministering the NgR1 polypeptides to mice or rats in models asdescribed herein.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof. Inorder that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

Example 1 Residues 15-28 in Aβ(1-28) are Essential for Binding to NgR

To determine whether a linear subsegment of Aβ(1-28) might interact withfull-length human NgR in a cell-binding assay, deletion constructscontaining various portions of the Aβ ectodomain fused to AP werecreated. AP-Aβ(1-28) protein was produced by the same method asAP-Nogo-66. To generate AP-Aβ mutant constructs, Aβ fragments wereamplified, ligated into the pAP5tag vector (GenHunter) and sequenced.Recoinbinant proteins were confirmed by immunoblotting. The binding ofAP fusion proteins to transfected COS-7 cells has been describedpreviously. Fournier et al., Nature 409:341-346 (2001). The region of Aβresponsible for full-length human NgR interaction localizes to residues15-28, the central residues of Aβ 1-40 (FIG. 1 a).

The binding of AP-Aβ(1-28) to NgR with that to other reported partners,p75 and RAGE was also compared. Deane et al., Nat Med 9:907-913 (2003);Yaar et al., J Clin Invest 100:2333-2340 (1997). COS-7 cells weretransfected with p75-NTR and RAGE, membrane proteins reported to bindAβ. Deane et al., Nat Med 9:907-913 (2003); Yaar et al., J Clin Invest100:2333-2340 (1997). Under conditions where AP-Aβ(1-28) binding to NgRis readily detectable, p75 and RAGE do not exhibit significantinteraction with Aβ fusion protein (FIG. 1 b).

NgR was identified by virtue of its affinity for Nogo-66, so weconsidered whether Aβ and Nogo-66 compete for binding to NgR.Competition was assessed in binding assays of AP-Aβ(1-28) orAP-Nogo66(1-33) to immobilized, purified NgR protein. Synthetic Aβ1-28was used to assess AP-AβB(1-28) and AP-Nogo-66(1-33) displacement fromimmobilized human NgR(310)ecto-Fc in an ELISA format. 250 nM solubleAP-Aβ(1-28) or AP-Nogo-66(1-33) was allowed to bind to wells coated withpurified NgR(310)ecto-Fc in the presence of the indicated concentrationsof free Aβ(1-28). In this cell-free assay, avidity for NgR is reducedcompare to the cell based binding system, and the measured K_(i) forAβ(1-28) is 700 nM. Data are means+/−SEM from 4 experiments. SyntheticAβ(1-28) disrupts NgR's ability to interact with the Aβ ligand but notthe Nogo-66 ligand (FIG. 1 c). Thus, the two ligand binding sites of NgRare distinguished by this assay.

Example 2 Specific Residues in NgR Support Binding to Ap-Aβ(1-28)

In order to probe the NgR domains that interact with Aβ and Nogo-66, astrategy based on the crystal structure of NgR was employed. A number ofhuman NgR surface-accessible residues were mutated to Ala eitherindividually or as groups of adjacent residues (Table 3), and resultantligand binding characteristics were assessed. The ligand concentrationswere AP, 30 nM, AP-Nogo-66, 5 nM, AP-Aβ(1-28), 50 nM. NgR mutagenesishas been previously described. Hu et al., J Neurosci 25:5298-5304(2005); Fournier et al., J Neurosci 23:1416-1423 (2003). The expressionof each mutant NgR protein was verified by immunohistochemical detectionat the surface of cells transfected with expression vector (FIG. 2 a).Bound AP was stained and measured using NIH image software. Mutants ofhuman NgR were detected on the surface of transfected COS-7immunofluorescently. For each of the mutants with altered bindingcharacteristics, expression of immunoreactive NgR protein withelectrophoretic mobility similar to wild type was also confirmed byimmunoblot (FIG. 2 c). Whole COS-7 cell lysates expressing NgR mutantswere subjected to SDS-PAGE and blotted with anti-NgR antibodies. Themobility of each mutant was indistinguishable from wild type NgR, exceptfor the mutations in N-linked glycosylation sites (N82 and N179). Thebinding of both AP-Aβ(1-28) and AP-Nogo-66 ligands to cells expressingthis collection of NgR mutant proteins was assessed at concentrationsequal to the predetermined Kd's of the ligands (FIG. 2 b, d).

Ala-substituted human NgR mutants were tested for their binding toAP-Aβ(1-28) and AP-Nogo-66. There are three categories: (a) NgR mutantsthat lose binding to both ligands, (b) mutants that maintain binding toall NgR ligands, and (c) differential binding mutants that bindAP-Nogo-66, but not AP-Aβ(1-28). A large group of amino acids areunnecessary for the binding of either ligand (Table 3). This includesall of the residues examined from the convex side of the NgR LRR domain.Another subset of amino acids are essential for the binding of bothAβ(1-28) and Nogo-66 (Table 3). Since these amino acids do not alter thelocalization or molecular size of NgR protein, and are clustered inclose proximity on the concave surface, we hypothesize that they form acore ligand binding site. This is consistent with the observation thatfor other LRR proteins, such as follicle-stimulating hormone receptor,ligand binding predominantly occurs on the concave side. Fan Q. R. andHendrickson W. A., Nature 433:269-277 (2005). Without structuralstudies, the possibility that these mutations prevent native NgR proteinfolding cannot be excluded. Most interesting are a third group of aminoacids, for which Ala substitution results in NgR binding of Nogo-66 butnot Aβ(1-28) (Table 3). Since AP-Nogo-66 binding is indistinguishablefrom wild type NgR, aberrant protein folding is unlikely to be the basisfor reduced Aβ binding. Instead, NgR amino acids 210, 256, 259 and 284are likely to contribute selectively to Aβ but not Nogo-66 interaction.

TABLE 3 Summary of human NgR mutants: list of residues mutated toalanine Binding to AP-Ng-66 and Differential No Binding AP-Aβ28 Binding163  61 210 82, 179  92 256, 259, 284 133, 136 108 158, 160 122 182, 186127 211, 213 131 232, 234 138 111, 113, 114 139 182, 186, 210 151 111,113, 114, 138 176 182, 186, 158, 160 179 189, 191, 211, 213 227 211,213, 237, 256, 259, 284 237 171, 172, 175, 176, 196, 199, 250 220, 223,224, 250 67, 68, 71 259 67, 68, 71, 89, 90, 92 108, 131 87, 89, 133, 136114, 117 Negative control 127, 151 127, 176 143, 144 189, 191 196, 199202, 205 256, 259 267, 269 277, 279 114, 117, 139 189, 191, 237 189,191, 284 202, 205, 227 202, 205, 250 220, 223, 224 237, 256, 259 296,297, 300 171, 172, 175, 176 292, 296, 297, 300 196, 199, 220, 223, 224171, 172, 175, 176, 196, 199 196, 199, 220, 223, 224, 250 108, 131, 6136, 38 36, 38, 61 61, 131, 36, 38 63, 65 78, 81 87, 89 89, 90, 114, 11795, 97 95, 97, 117, 119, 120, 188, 189 95, 97, 122 Wild type

Example 3 NgR(310)ecto-Fc Treatment Acts Peripherally to Alter thePlasma/Brain Aβ Ratio

While endogenous NgR plays a role in limiting Aβ production anddeposition, the affinity of NgR for the central domain of Aβ suggeststhat it might promote peripheral clearance if delivered outside of theCNS. To examine whether rat NgR(310)ecto-Fc administered subcutaneouslyenters the brain of mouse, the presence of NgR(310)ecto-Fc in brainlysates was assayed. The NgR(310)ecto-Fc fusion protein or control ratIG was concentrated by protein A/G affinity chromatography. Toadminister rat NgR(310)ecto-Fc protein, APPswe/presenilin-1 (Psen-1)ΔE9mice (Park et al., J Neurosci 26:1386-1395 (2006)) from JacksonLaboratories (Bar Harbor, Me.) (Stock #04462) were anesthetized withisoflurane and oxygen and an ALZET osmotic pump 2004 was subcutaneouslyinserted over the scapula and allowed to rest between fascia. The pumpdelivered 0.25 μl/hr for 28 days of a 1.2 μg/μl solution of ratNgR(310)ecto-Fc or rat IgG in PBS. Pumps were replaced after 28 days fortotal treatment duration of 12 weeks. The anti-Aβ (6E10) antibody wasfrom Chemicon. DAB staining reagents were from Vector. The dose of eachprotein was 0.27 mg/kg/d.

Brains from subcutaneously treated APPswe/Psen-1ΔE9 transgenic mice werehomogenized in PBS plus Protease Inhibitor Cocktail (Roche). Theparticulate fractions were collected by centrifugation at 100,000×g for20 min. Membranes were resuspended in PBS (1 gm brain wet weight/ml) andsolubilized in 1% Triton X-100. The detergent extract was subjected toProtein A/G Plus Sepharose (Pierce, Rockford, Ill.) immunoprecipitationand analyzed by anti-NgR polyclonal antibody from R&D Systems, Inc.(AF1440). While intracerebroventricular administration leads to easilydetected NgR(310)ecto-Fc levels in brain tissue, no NgR(310)ecto-Fc isdetected centrally after subcutaneous treatment (FIG. 3 a). This isconsistent with the hypothesis that NgR(310)ecto-Fc cannot pass the BBBto an appreciable degree in APPswe/Psen-1ΔE9 mice.

To the extent that NgR(310)ecto-Fc functions as a peripheral sink forAβ, the ratio of plasma to brain Aβ should be elevated, as shown foranti-Aβ treatment. Levels of Aβ40 and Aβ42 were assessed byenzyme-linked immunosorbent assay (ELISA) in brain and plasma samplesfrom peripherally treated mice (FIG. 3 b). After three months ofsubcutaneous treatment, there is a significant increase in theplasma:brain Aβ(1-42) ratio, *, p<0.05, ANOVA. Aβ ELISA assays wereperformed according to manufacturer's protocol (Biosource, Inc).Subcutaneous treatment with NgR(310)ecto-Fc increased plasma:brainratios for Aβ more than two-fold. Previously, we noted that central,i.c.v.-administered NgR(310)ecto-Fc reduces levels of sAPPα and sAPPβprotein in the brain. Park et al., J Neurosci 26:1386-1395 (2006).However, brain APP levels are not altered by subcutaneousNgR(310)ecto-Fc treatment (FIG. 3 c, d). Mean±sem from n=4-5 mice.

Example 4 Reduction of Aβ Plaque Load, Neuritic Dystrophy, andAstrocytosis in Ngr(310)ecto-Fc-Treated APPswe/PSEN-1ΔE9 Mice

The restriction of subcutaneous NgR(310)ecto-Fc to the periphery allowsan assessment of its effect as a “sink” on central Aβ burden. Treatmentof APPswe/PS-1ΔE9 transgenic mice was initiated at 7 months of age whenthe mice have become symptomatic, as judged by Aβ deposition in brainand by reduced spatial memory function (see below). After 3 months oftreatment with 0.27 mg/kg/day of subcutaneous NgR(310)ecto-Fc versus IgG(0.6 mg of total protein), the brain was examined byimmunohistochemistry and ELISA. Aβ plaques in parasagittal sections werefixed by paraformaldehyde and labeled with anti-Aβ-(1-17) 6E10 antibodyafter 0.1 M formic acid treatment. Plaque area was quantitated using NIHImage as a percentage of total cerebral cortical area for two sectionsfrom each animal. Neuritic dystrophy and reactive astrocytosis werevisualized by staining with monoclonal anti-synaptophysin GA-5 (Sigma)and monoclonal anti-GFAP SY 38 (Chemicon) in parasagittalparaffin-embedded sections. The area of cerebral cortex and hippocampusoccupied by clusters of dystrophic neurites and reactive astrocytes weremeasured as a percentage of total area by the same method as Aβ plaqueload. Data are ±SEM from 9 mice in rat IgG treated group and 7 mice fromNgR(310)ecto-Fc treated group.

The total Aβ(1-40) and Aβ(1-42) levels as well as Aβ plaque aredecreased significantly by NgR(310)ecto-Fc, to a level approximately 50%of control (FIG. 4 a, d, e). In parallel, dystrophic neurites detectedby anti-synaptophysin staining are decreased by peripheralNgR(310)ecto-Fc treatment (FIG. 4 b, f). Astrogliosis detected byanti-GFAP staining intensity was also reduced significantly by therapywith peripheral NgR(310)ecto-Fc (FIG. 4 c, g). Thus, delayedsubcutaneous administration of NgR(310)ecto-Fc suppresses histologicevidence of Aβ-associated disease in transgenic mice.

Example 5 Subcutaneous Treatment of NgR(310)ecto-Fc Improves Radial ArmWater Maze Performance in APPswe/PSEN-1ΔE9 Transgenic Mice

The ability of subcutaneous NgR(310)ecto-Fc therapy to reduce Aβ plaqueis encouraging, but cognitive performance is the relevant symptom inclinical AD. To assess APPswe/PS-1ΔE9 transgene-related impairments inspatial memory, a modified radial arm water maze paradigm (RAWM) wasemployed. Morgan et al., Nature 408:982-985 (2000). A modified radialarm water maze testing protocol was based on personal communication withD. Morgan (Morgan et al., Nature 408:982-985 (2000)). The maze consistedof a circular pool one meter in diameter with six swim alleys nineteencm wide that radiated out from a 40 cm open central area and a submergedescape platform was located at the end of one arm. Spatial cues werepresented on the walls and at the end of each arm. The behaviorist wasblind to treatment. To control for vision, motivation and swimming, micewere tested in an open water visual platform paradigm for up to oneminute and latency times were recorded. Next, mice were placed in arandom arm according to an Excel function=MOD($CELL+RANDBETWEEN(1,5),6),where $CELL is the location of the hidden platform. Each mouse wasallowed to swim up to one minute to find the escape platform. Uponentering an incorrect arm (all four paws within that swim alley) orfailing to select an arm after twenty seconds, the mouse was pulled backto the start arm and charged an error. All mice spent 30 seconds on theplatform following each trial before beginning the next trial.Thereafter, the mouse was tested four more times, constituting alearning block. Mice were allowed to rest for 30 minutes betweenlearning blocks. In total, mice were tested over three learning blocksover the first day and on the following day another three learningblocks were repeated.

Short-term spatial memory deficits are apparent in APPswe/PS-1ΔE9 versuswild type littermate mice by 4 months (FIG. 5 a). By 13 months of age,wild type mice perform less well at this task than do young mice, whileAPPswe/PS-1ΔE9 transgenic mice are completely unable to learn the taskin our training paradigm illustrating disease progression (FIG. 5 b). Asa control, loss of NgR expression (in ngr−/− mice) does notsignificantly alter RAWM performance (FIG. 5 c).

The number of swim errors made by APPswe/PS-1ΔE9 mice after 25-29training trials increases steadily at 8, 9 and 10 months when micereceive control IgG therapy subcutaneously for 1, 2 or 3 months. Incontrast, mice treated with subcutaneous NgR(310)ecto-Fc exhibit a haltin disease progression, and show a trend towards improved performanceafter 3 months, by 10 months of age (FIG. 5 d). RAWM errors aresignificantly reduced after two months and after three months ofsubcutaneous NgR(310)ecto-Fc treatment compared to rIgG-treated mice(ANOVA, P<0.05 and 0.02, respectively). These differences are related toimproved memory function rather than altered vision, motivation or motorcapacity, since no significant difference was observed in visibleplatform escape latencies between these groups (FIG. 6). Mean±sem fromn=7-9 mice per group. There is a positive correlation between theaverage RAWM errors and the density of Aβ-immunoreactive deposits acrossthe two groups (FIG. 5 e).

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

1. A method of increasing the plasma to brain ratio of Aβ peptide in amammal, comprising administering to a mammal in need thereof atherapeutically effective amount of a soluble Nogo receptor polypeptide,wherein said administration is peripheral to the central nervous system.2. A method of enhancing Aβ clearance from the brain of a mammal,comprising administering to a mammal in need thereof a therapeuticallyeffective amount of a soluble Nogo receptor polypeptide, wherein saidadministration is peripheral to the central nervous system.
 3. A methodof improving memory function or inhibiting memory loss in a mammalcomprising administering to a mammal in need thereof a therapeuticallyeffective amount of a soluble Nogo receptor polypeptide, wherein saidadministration is peripheral to the central nervous system.
 4. A methodof reducing the number of Aβ plaques in the brain of a mammal,comprising administering to a mammal in need thereof a therapeuticallyeffective amount of a soluble Nogo receptor polypeptide, wherein saidadministration is peripheral to the central nervous system.
 5. A methodof reducing the size of Aβ plaques in the brain of a mammal, comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of a soluble Nogo receptor polypeptide, wherein saidadministration is peripheral to the central nervous system.
 6. A methodof treating a disease associated with Aβ plaque accumulation in a mammalcomprising administering to a mammal in need thereof a therapeuticallyeffective amount of a soluble Nogo receptor polypeptide, wherein saidadministration is peripheral to the central nervous system.
 7. Themethod of claim 6, wherein said disease is selected from the groupconsisting of Alzheimer's disease, mild cognitive impairment,mild-to-moderate cognitive impairment, vascular dementia, cerebralamyloid angiopathy, hereditary cerebral hemorrhage, senile dementia,Down's syndrome, inclusion body myositis, age-related maculardegeneration, primary amyloidosis, secondary amyloidosis and a conditionassociated with Alzheimer's disease.
 8. The method of claim 7, whereinsaid condition associated with Alzheimer's disease is selected from thegroup consisting of hypothyroidism, cerebrovascular disease,cardiovascular disease, memory loss, anxiety, a behavioral dysfunction,a neurological condition, and a psychological condition. 9-11.(canceled)
 12. The method of claim 1, wherein said mammal is a human.13. The method of claim 1, wherein said soluble Nogo receptorpolypeptide is administered subcutaneously, parenteraly parenterally,intravenously, intramuscularly, intraperitoneally, transdermally,inhalationaly or buccally.
 14. The method of claim 1, wherein saidsoluble NgR1 polypeptide is 90% identical to a reference amino acidsequence is selected from the group consisting of: (i) amino acids 27 to310 of SEQ ID NO:2; (ii) amino acids 27 to 344 of SEQ ID NO:2; (iii)amino acids 27 to 445 of SEQ ID NO:2; (iv) amino acids 27 to 309 of SEQID NO:2; (v) amino acids 1 to 310 of SEQ ID NO:2; (vi) amino acids 1 to344 of SEQ ID NO:2; (vii) amino acids 1 to 445 of SEQ ID NO:2; (viii)amino acids 1 to 309 of SEQ ID NO:2; (ix) variants or derivatives of anyof said reference amino acid sequences, and (x) a combination of one ormore of said reference amino acid sequences or variants or derivativesthereof.
 15. The method of claim 14, wherein said soluble NgR1polypeptide is selected from the group consisting of: (i) amino acids 27to 310 of SEQ ID NO:2; (ii) amino acids 27 to 344 of SEQ ID NO:2; (iii)amino acids 27 to 445 of SEQ ID NO:2; (iv) amino acids 27 to 309 of SEQID NO:2; (v) amino acids 1 to 310 of SEQ ID NO:2; (vi) amino acids 1 to344 of SEQ ID NO:2; (vii) amino acids 1 to 445 of SEQ ID NO:2; (viii)amino acids 1 to 309 of SEQ ID NO:2; (ix) variants or derivatives of anyof said polypeptides; and (x) a combination of one or more of saidpolypeptides or variants or derivatives thereof. 16-23. (canceled) 24.The method of claim 1, wherein said soluble Nogo receptor polypeptidecomprises a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to twenty individual amino acid substitutions, wherein said firstreference amino acid sequence is selected from the group consisting of:(a) amino acids a to 445 of SEQ ID NO:2, (b) amino acids 27 to b of SEQID NO:2, and (c) amino acids a to b of SEQ ID NO:2, wherein a is anyinteger from 25 to 35, and b is any integer from 300 to 450; and whereinsaid second polypeptide fragment comprises an amino acid sequenceidentical to a second reference amino acid sequence, except for up totwenty individual amino acid substitutions, wherein said secondreference amino acid sequence is selected from the group consisting of:(a) amino acids c to 445 of SEQ ID NO:2, (b) amino acids 27 to d of SEQID NO:2, and (c) amino acids c to d of SEQ ID NO:2, wherein c is anyinteger from 25 to 35, and d is any integer from 300 to
 450. 25-27.(canceled)
 28. The method of claim 14, wherein at least one amino acidresidue of said soluble NgR1 polypeptide is substituted with a differentamino acid.
 29. (canceled)
 30. (canceled)
 31. The method of claim 14,wherein said soluble NgR1 polypeptide is a cyclic polypeptide. 32-36.(canceled)
 37. The method of claim 14, wherein said soluble NgR1polypeptide further comprises a non-NgR1 moiety.
 38. The method of claim37, wherein said non-NgR1 moiety is a heterologous polypeptide fused tosaid soluble NgR1 polypeptide. 39-44. (canceled)
 45. The method of claim37, wherein said soluble NgR1 polypeptide is conjugated to a polymer.46-50. (canceled)
 51. The method of claim 1, wherein the therapeuticallyeffective amount is from 0.001 mg/kg to 10 mg/kg of soluble Nogoreceptor polypeptide.
 52. (canceled)
 53. (canceled)
 54. The method ofclaim 1, wherein the soluble Nogo receptor polypeptide does not crossthe blood-brain barrier.
 55. The method of claim 1, wherein said solubleNogo receptor polypeptide is coadministered with one or more anti-Aβantibodies.
 56. The method of claim 55, wherein said soluble Nogoreceptor polypeptide is coadministered one or more additionaltherapeutic agents, selected from the group consisting of an adrenergicagent, anti-adrenergic agent, anti-androgen agent, anti-anginal agent,anti-anxiety agent, anticonvulsant agent, antidepressant agent,anti-epileptic agent, antihyperlipidemic agent, antihyperlipoproteinemicagent, antihypertensive agent, anti-inflammatory agent, antiobessionalagent, antiparkinsonian agent, antipsychotic agent, adrenocorticalsteroid; adrenocortical suppressant; aldosterone antagonist; amino acid;anabolic steroid; analeptic agent; androgen; blood glucose regulator;cardioprotectant agent; cardiovascular agent; cholinergic agonist orantagonist; cholinesterase deactivator or inhibitor, cognition adjuvantor enhancer; dopaminergic agent; enzyme inhibitor, estrogen, free oxygenradical scavenger; GABA agonist; glutamate antagonist; hormone;hypocholesterolemic agent; hypolipidemic agent; hypotensive agent;immunizing agent; immunostimulant agent; monoamine oxidase inhibitor,neuroprotective agent; NMDA antagonist; AMPA antagonist, competitive or-non-competitive NMDA antagonist; opioid antagonist; potassium channelopener; non-hormonal sterol derivative; post-stroke and post-head traumatreatment; prostaglandin agent; psychotropic agent; relaxant agent;sedative agent; sedative-hypnotic agent; selective adenosine antagonist;serotonin antagonist; serotonin inhibitor; selective serotonin uptakeinhibitor; serotonin receptor antagonist; sodium and calcium channelblocker; steroid; stimulant; and thyroid hormone and inhibitor agents.